JPS60224636A - Production of antibody of high quality - Google Patents

Abstract

PURPOSE:To obtain an antibody of high quality having higher affinity and specificity than those of a raw material antibody which is a polyclonal antibody, by isoelectric focusing of the raw material antibody. CONSTITUTION:A raw material antibody collected by giving an antigen to immunize an animal is subjected to isoelectric focusing to afford the aimed antibody having higher affinity and specificity than those of the raw material antibody which is not a simple antibody but an aggregate of many immunoglobulins having various specificities and affinities. So far as the raw material antibody is used as a mixture, averaged rather low specificity and affinity are produced. The isoelectric focusing is carried out by dispersing various amphoteric carriers having individual isoelectric points in a floating field, placing a spot of the raw material antibody at an optional point, applying electric charge thereto, moving the above-mentioned antibody to the positions of the respective intrinsic isoelectric points, and separating the antibody as a mixture or simple substance having the isoelectric points as a common factor. The simple substance exhibits higher affinity and specificity than those of the raw material antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for collecting high quality antibodies from a large number of cloned antibodies by utilizing the technique of isoelectric focusing.

[Conventional technology]

An animal given an antigen acquires immunity, and the antiserum collected from the animal contains antibodies that specifically react with the antigen. However, this antibody is not a single antibody, but a collection of many immunoglobulins (hereinafter referred to as Ig) with various specificities and affinities, and is generally called a polyclonal antibody. I'm here. In other words, polyclonal antibodies can be said to be a collection of various monoclonal antibodies with different specificities and affinities.

On the other hand, the specificity of antigen-antibody reactions (immunological reactions) can be reproduced not only in vivo but also in vitro, so for example, it is possible to identify a target antigen from a specimen containing a mixture of various substances (antigens). This antigen-antibody reaction is used for detection, identification, or quantification. Therefore, if you are going to apply an antigen-antibody reaction for analysis, it is desirable to use high-quality antibodies with high specificity and high affinity.It is said that the quality of specificity and affinity determines the quality of the results. ing.

However, as mentioned earlier, the antibodies in antiserum are polyclonal antibodies, and are a mixture of antibodies with high specificity and affinity and antibodies with low specificity and affinity, so these should be used as a mixture. Only a limited, averaged and lower specificity or affinity is expressed. Therefore, efforts have been made to obtain high-quality antisera by: ■ using sufficiently purified antigens for immunization injections, ■ selecting appropriate antigens for immunization injection animals, and ■ devising immunization injection methods. ing. Although these methods can sometimes yield products with high potency, specificity, and affinity, the results are not consistent and may vary due to individual differences among animals. It is not always possible to obtain high quality antibodies.

From this point of view, a method of improving affinity by using actually obtained polyclonal antibodies as raw materials and removing endogenous antigens bound to the antibodies,
Furthermore, a method of extracting highly specific monogenic antibodies using the gun fusion method is being studied. However, the former method does not necessarily yield consistent results, and the latter method requires the complexity of handling a large number of living cells, as well as the need to screen each one individually. However, the fact is that the monoclonal antibodies that are finally obtained by this process often have low affinity.

[Problem that the invention seeks to solve]

Concerned about this situation, the present inventors used polyclonal antibodies obtained based on general methods as raw materials, and selected monoclones with excellent specificity and affinity (especially affinity) from among the raw antibodies. We have started investigating methods for extracting sexual antibodies. In particular, the present inventors have discovered that the amino acid composition and amino acid sequence constituting Ig differ slightly depending on each monoclonal antibody, and that due to these differences, each antibody has its own unique isoelectric point (pI). By focusing on the fact that
Moreover, because the separated substances are collected singly or as a mixture with the isoelectric point as a common factor, they may have unique chemical properties as antibodies, especially in terms of affinity and specificity. After many basic experiments, they finally completed the invention.

[Means for solving problems]

The gist of the present invention is to give an antigen to an animal for immunization.The collected polyclonal antibody is used as a raw material, and by subjecting it to isoelectric focusing, the raw material antibody can also be immunized. It exists in the collection of both types of antibodies that exhibit high affinity and high specificity characteristics.

[Effect]

Isoelectric focusing is a method in which various amphoteric carriers, each with its own isoelectric point, are dispersed in the electrophoresis field to which a charge is to be applied, and the raw material polyamide is placed at any point in the electrophoresis field. A method of placing a spot of clonal antibody and applying a charge,
Since the amphoteric carriers exhibit different pH values depending on their respective isoelectric points, a pH gradient is formed along the direction of potential difference. Therefore, when each mono-quaternary antibody in the spotted raw material antibody moves toward its unique isoelectric point position, it is separated as a mixture or a single substance with the isoelectric point as a common factor. Ru. Therefore, mixtures or single substances separated by electrochemical properties exhibit their respective chemical properties as antibodies, particularly in terms of affinity and specificity.

Supports used in the isoelectric focusing of the present invention include polyacrylamide gels, agarose gels, Sephadex gels, etc., but of course there is no reason to limit them to these. There are no restrictions on the type or number of amphoteric carriers or the means for forming an electric field.

Furthermore, the raw material antibodies to which the present invention is applied are not limited at all, and it does not depend on what kind of antigen is given to what kind of animal and how it is immunized, and any polyclonal antibody can be used. It can be used in the present invention. However, the manner of separation differs somewhat depending on the antibody, and there are cases in which an antibody with particularly high affinity is obtained, and there are cases in which an antibody with excellent not only affinity but also high specificity is obtained. For example, antiserum obtained by immunizing a rabbit with human thyroid stimulating hormone (hTSH) (
The immunoglobulin G (IgG) fraction of hTsH antiserum was subjected to isoelectric focusing using an agarose gel as a support.
When separated using IEF), it can be divided into more than 20 types of protein bands, some of which are highly specific and high affinity anti-hTSH antibodies. According to research on the separation mechanism in this example, (1) the Fc7 fragment obtained by pepsin treatment of IgG was separated into two protein bands by agarose gel IEF at 9 pH; between 7.0 and 7.3. (2) The F (a b')1 fragment (containing the antigen-binding site) obtained in the same manner was found to contain many proteins with h, p I: between 5 and 90. Combining the results of (1) and (2), the separation of polyclonal rabbit IgG by IEF is based on the pI of the F(ab')17;7 segment. It can be concluded that there is.

In any case, in the present invention, antibodies with particularly high affinity or high specificity may be collected from the protein bands thus separated.

〔Example〕

Example 1 Isolation of highly specific and high affinity anti-hTSH antibodies ■ Method Using anti-hTsH rabbit serum, IgG was isolated by salting out with sodium sulfate and diethylamine ethylcellulose, or by performing four-mardography. A sample was prepared (anti-hTSHIgG) and subjected to isoelectric focusing below, and the asterisk (*) indicates that a product manufactured by Pharmacia was used. Agarose gel sprays (12×12 cm) containing Pharmalyte at pH ranges of 3-10 and 5-8 were prepared. The electrode solution is IN sodium hydroxide aqueous solution on the cathode side and 0.05M on the anode side.
It was made into sulfuric acid.

Place the agarose gel plate in a flatbed electrophoresis device (FBE-3000/150), and place it in a constant voltage device (E
CPS-3000/150W') at 8W under cooling.
．． Electricity was applied until it reached 7KV. Anti-hTsHIgG (10-20μl) after dialysis against 8mM sodium chloride aqueous solution
) was spotted on the filter paper at a position 3 to 4 cm from the cathode side (for protein staining, 0.1 to 0.2 caps of IgG was spotted at 2 cm from the cathode side).
Uo, 4-1 for recovery after IEF separation.
1 ng of IgG was added to 4-5 filter papers). First 1
．． A current of 8 W was applied for about 60 minutes until a voltage of 5 KV was obtained, and then a current of 1.5 KV was applied for 30 to 60 minutes. After that, the plastic gel plate was divided into two parts, and one part was used for tanno(reactive staining) and the other part was used for antibody recovery.For protein staining, first, the plastic gel plate was divided into two parts, and the other part was used for antibody recovery. The proteins were fixed in the gel by soaking for 30 minutes, and then washed with a 30% ethanol solution containing 1° vinegar and stained with 0.2 ml Brilliant Blue R.

Meanwhile, for antibody recovery from the gel, cut the gel on the divided plate into 2 mm width pieces, transfer each piece to a test tube containing 0.1% bovine serum albumin and 0.1% NaN6. This was carried out by adding 0.1 M phosphate buffer, pH 8.0 (buffer T), and leaving the mixture at 4° C. for over a day and night.

The extract was then collected, and the antibody titer, binding affinity and specificity for hTsH were determined by radioimmunoassay (RIA).
). This was determined from the pH gradient on the gel, the pI property of the separated antibody molecules, and the mobility of the standard protein y from the pI measurement kit, which was electrophoresed in the same manner. In RIA for measuring antibody titer and binding affinity, first, each antibody fraction before and after IEF separation is subjected to approximately 8 nCI of "I-" in the presence (or absence) of various concentrations of unlabeled hTSH. hT
SH and incubated in buffer T in a final volume of 300 pl. The amount of antibody used here was adjusted so that the amount of antibody binding to I-hTSH was approximately 50%. After incubating under these conditions at room temperature for 20 hours, 4 μl of normal rabbit serum and an optimal amount of anti-rabbit 1gG goat serum were added, and the mixture was incubated again overnight in an incubation state, centrifuged at 2000 g for 15 minutes, and the precipitate was collected by r- Measured with a counter. The binding affinity constant was calculated by analysis using 5catchard plot. The binding affinity for hTSH was determined by the excess amount of human placental gonadotropin (hCG) (1, I
) Measurements were made in the same manner in the presence of The specificity of the antibody to hTSH was examined using cross-reactivity to hCG as an index. That is, each antibody fraction was treated with various concentrations of hC.
incubating with 1251-hTSH in the presence of G;
The extent of replacement of hTSH by hCG was determined by a similar RIA method.

■Results pI of each antibody fraction separated by IEF in the pH range of 3-10 and 5-8, cross-reactivity with hCG, hTSH
The binding affinity with is shown in Table 1.

When IEF was performed in the pH range of 3 to 10, an antibody with less cross-reactivity to hCG, that is, with high specificity, was obtained almost in parallel with the decrease in pl. Similar results regarding cross-reactivity were also obtained when IEF was performed in the pH range of 5 to 8, but antibodies obtained from fraction numbers 11 and 21 rarely showed cross-reactivity (cross-reactivity The rate is approximately 1/2 that of the original serum).

Furthermore, the binding affinity constant of fraction number 13 was approximately twice that of the original serum in the absence of hCG.

Example 2 Isolation of highly affinity anti-thyroxine (T4) antibody Method Preparation of an IgG fraction from anti-T4 rabbit serum and separation and recovery of the IgG fraction using agarose IEF were carried out in the same manner as for the anti-hTSH antibody. However, for extraction of antibodies from agarose gel strips, 20mM) Lis-HCl buffer containing ~40μg of 8-anilino-1-naphthalene sulfate and 1.67ay/g of salicylic acid, pH 9.0 (Buffer R), is used.
was used.

In RIA, which measures antibody titer and binding affinity,
Each antibody fraction was incubated at approximately 8 nci in 300 μm of Buffer R.
(Incubated with D 126I-74. After approximately 20 hours of incubation at room temperature, manipulation and analysis were carried out with anti-h
The same procedure as for the TSH antibody was carried out.

■Results T of antibodies fractionated by IEF in the pH range of 3 to 10
The binding affinity constant for , was in the range of (1,8X 10'') 1 to (8,3X10') M. The highest value among these was the binding affinity constant Z, 2 of the raw serum (IgG fraction). x 10'M-'.
In IA, a measurement sensitivity 3 to 5 times higher was obtained.

〔Effect of the invention〕

Since the present invention is configured as described above, a polyclonal antibody obtained by a conventional method is used as a raw material, and a high-quality antibody with particularly high affinity and specificity compared to the raw material antibody is produced by a relatively simple method. It became possible.

Claims (1)

[Claims] It is characterized by collecting raw antibodies (by giving antigen to animals for immunization) and subjecting them to isoelectric focusing to collect antibodies with high affinity and specificity (as compared to raw antibodies). A method for producing high-quality antibodies.