JP2508080B2 - Method for purifying anti-steroid antibody - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for purifying an anti-steroid antibody used in an immunoassay method.

(Prior Art) In recent years, a so-called immunoassay method using an antibody has come to be widely used as a means for measuring a trace component in a body fluid. In this measuring method, obtaining an antibody with high specificity for an antigen is an important point for increasing the measurement sensitivity.

In general, antibodies against low molecular weight substances such as steroids
A low molecular weight substance is bound to a carrier protein such as bovine serum albumin and sensitized with an animal such as a rabbit. The antibody thus obtained is a so-called polyclonal antibody, and is considered to be a mixture of antibodies that recognize various sites of the low-molecular-carrier protein conjugate sensitized as an antigen.

On the other hand, among the immunoassay methods, the enzyme immunoassay method is an excellent detection method in that it has high sensitivity and there is no risk of contamination. By the way, the enzyme immunoassay method for low-molecular substances is based on the principle of competition between the oxygen-binding low-molecular antigen (hereinafter referred to as conjugate) in the binding reaction with the antibody and the antigen in the sample. However, in the case of a connecting portion (spacer portion) between the low molecular weight antigen and the carrier-protein, the antibody strongly binds to the conjugate, and as a result, sufficient measurement sensitivity cannot be obtained.

There are the following two types of methods that are generally used to deal with this. The first method is to make the binding site between the low molecular weight substance and the carrier protein in the sensitized antigen the same as the binding site between the enzyme of the conjugate and the low molecular weight substance (site heterologous assay). The second method is to change the structure of the spacer between the conjugate and the substance to be sensitized (bridge heterologous, assay) (problems to be solved by the invention) However, depending on the type of antibody or small molecule antigen, The measurement sensitivity may not be improved even if such a method is used. Such an antibody could not be used for enzyme immunoassay.

(Means for Solving Problems) As a result of various studies on methods for improving the measurement sensitivity by using a combination of an antibody and a conjugate, which cannot obtain sufficient measurement sensitivity in the prior art, we strongly recognize the spacer portion. This object can be achieved by the operation of removing the antibody, and the present invention has been completed.

That is, an antibody mixture prepared by sensitizing a mammal with a steroid-modified protein consisting of a protein bound to a steroid through a spacer is used, and different steroids having the same or similar steroid skeleton structure as those used for the antibody preparation are used. A steroid-modified protein bound to the same protein via a spacer, wherein at least the binding position between the spacer and the steroid is the same as the binding position between the spacer and the steroid in the steroid-modified protein used during antibody production, The present invention relates to a method for purifying an anti-steroid antibody, which comprises contacting with an immobilized water-insoluble carrier for purification.

Hereinafter, the present invention will be described in detail.

The steroid bound to the protein and immobilized on the carrier is not particularly limited as long as it is different from the steroid used to prepare the anti-steroid antibody, but preferably,
Binding of steroids to proteins A substance having a ring structure (eg, A ring, B ring, etc.) of the same structure as or similar to the steroid antigen is preferable. Further, the structures of the spacers are the same, and at least the binding positions of the steroid and the spacer are the same among the binding positions of the protein and the steroid. Here, the purpose of preliminarily binding the steroid to the protein at the time of immobilization is to adsorb an antibody that recognizes across the spacer, the protein, or both, and in this sense, the protein used for immobilization is the antigen protein. It is preferable that they are the same.

The protein used in the present invention may be one that can be recognized as an antigen in a living body, and its molecular weight is preferably 10,000 or more, more preferably 20,000 to 100,000. An example of this is bovine serum albumin (BS
A), human serum albumin (HSA), thyroglobulin and the like can be mentioned, but BSA is preferred from the viewpoint of easy availability.

As a water-insoluble carrier, --NH ₂ , --OH, Any functional group which has a functional group and can form a covalent bond with a protein by a chemical treatment such as a bromocyanogen method or a carbodiimide method. Examples thereof include natural polymers such as crosslinked dextran, crosslinked agarose, and cellulose, acrylic acid polymers such as glycidyl methacrylate and hydroxyethyl acrylate, crosslinked polyvinyl alcohol, ethylene-vinyl acetate copolymer, and crosslinked polyacrylamide. Polymers can be mentioned. The particle size is 10 to 1000 μm, preferably 50 to 200 μm
Is good.

The amount of the steroid-protein immobilized on the water-insoluble carrier is -0.01 to 10% of the dry weight of the water-insoluble carrier, preferably 0.
1-2% is good.

Steroids used in the present invention include progesterone, cortisone, cortisol, corticosterone, testosterone, aldosterone, estrone, estradiol-17β, estriol, estradiol-
3-sulfate, 17α-hydroxyprogesterone,
Pregnane diol etc. can be mentioned. In particular, when the steroid antigen is progesterone-3-carbomethyloxime-BSA (progesterone-3CMO-BSA), cortisol-3CMO-BSA, testosterone-3CMO-BSA, or aldosterone-3CMO-BSA is bound to a carrier to form a steroid. When the antigen is 11α-hydroxytestosterone-11 hemisuccinate-BSA, 11α-hydroxyprogesterone-11 hemisuccinate-BSA or 11 epicortisol-11 hemisuccinate-BSA is bound to a carrier, and the steroid antigen is cortisol-21 hemisuccinate-BS.
In the case of A, cortisol-3 sulphate-21 hemisuccinate-BSA or tetrahydrodeoxycorticosterone-21 hemisuccinate-BSA is bound to a carrier, and when the steroid antigen is 6oxoestriol-6CMO-BSA, 6oxoestradiol-3 Salifate-6C
It is desirable to attach MO-BSA to a carrier and subject it to the method of the present invention. Further, even if the combination of these steroids is reversed, it is effective for the method of the present invention.

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited thereto.

(Example) Preparation of antibody Rabbits were immunized with commercially available progesterone-3CMO-BSA (manufactured by Sigma), and IgG was purified from the obtained serum. The IgG fraction thus obtained was mixed with cross-linked and insolubilized BSA to prepare BS.
After removing the antibody against A, it was used for the following purification. The antibody did not have cross-reactivity with cortisol.

Preparation of the carrier 85 mg cortisol-3-carboxymethyl oxime was combined with 100 mg BSA by the carbodiimide method. 10 ml of this cortisol-3-CMO-BSA solution (4.3 mg in BSA concentration)
/ ml) suspended in 50 mM sodium carbonate buffer pH 8.3
Br-activated Sepharose ”(trade name, manufactured by Pharmacia Fine Chemicals) was mixed and reacted for 4 hours. After completion of the reaction, the remaining active groups were blocked with a 100 mM monoethanolamine aqueous solution.

The amount immobilized on Sepharose was about 1 wt% (based on the dry weight of Sepharose).

Purification of antibody 2.5 ml of cortisol-3-carpoxymethyloxime-BSA Sepharose prepared as described above was packed in a column, and 3 ml of the antibody solution was slowly passed through. The IgG in the non-adsorbed fraction was immobilized on a microtiter plate, and the following enzyme immunoassay was performed.

Oxygen immunoassay cortisol-3-carboxymethyl oxime-BSA
Antibody purified on Sepharose column and as a control experiment
Enzyme-linked immunosorbent assay was performed on an antibody-immobilized microplate treated with a BSA-immobilized column. The results are shown in FIG.

The sample used was serum obtained by removing steroids from charcoal powder with the indicated amount of progesterone added, and as the conjugate, progesterone-3-carboxymethyloxime-alkaline phosphatase was used. After completion of the antigen-antibody reaction, the plate was washed, p-nitrophenyl phosphate was added as an enzyme substrate, and the absorbance at 405 nm was measured.

In FIG. 1, Bo represents the absorbance when progesterone was not added, and B represents the absorbance when a predetermined amount of progesterone was added. As is clear from FIG. 1, the measurement sensitivity was significantly increased by the purification method of the present invention.

(Effect of the invention) By using the purification method of the present invention, an antibody that strongly recognizes the spacer portion can be removed, and therefore the measurement sensitivity in the enzyme immunoassay is improved. Further, the method of the present invention is effective when the enzyme immunoassay method must be applied when there is no method for changing the type of spacer or it is difficult. Furthermore, the protein-bound antigen and the enzyme-bound antigen can be prepared by using only one type of antigen-binding method, which provides a convenient method for the experimenter.

[Brief description of drawings]

FIG. 1 is a diagram showing the absorbance of an antibody purified according to the method of the present invention.

Claims (1)

(57) [Claims] 1. An antibody mixture prepared by sensitizing a mammal with a steroid-modified protein consisting of a protein bound to a steroid through a spacer, and having a steroid skeleton structure different from or the same as that used in the antibody preparation. A steroid-modified protein in which a steroid is bound to the same protein through the same spacer, and at least the binding position between the spacer and the steroid is the same as the binding position between the spacer and the steroid in the steroid-modified protein used during antibody production. A method for purifying an anti-steroid antibody, which comprises contacting a protein with an immobilized water-insoluble carrier for purification.