JPH05105700A - Serum or antibody resistant to yeast, their preparation and their employment - Google Patents

Abstract

PURPOSE:To obtain the subject antibody used for the purification of a B type hepatitis vaccine produced from a yeast as a host by immunizing a vertebrate with the yeast or its fragment to prepare an astisera recognizing the yeast, and subsequently purifying the antisera to give an immunoglobulin fraction. CONSTITUTION:A yeast (e.g. Pichia pastoris) or its fragment as an antigen is intravenously injected into a vertebrate (e.g. rabbit) by the use of an injector for the immunization of the vertebrate. The same operation are repeated every three days after the first injection, and after 24 days, the whole blood of the rabbit is collected. The blood is centrifuged to give the serum, from which an antisera recognizing the yeast is prepared. The antisera is subjected to an ammonium sulfate fractionation treatment, and the obtained IgG fraction (immunoglobulin G) is applied to a column filled with a carrier bonded to protein A, etc. The adsorbed fraction is eluted to obtain the objective antibody specifically recognizing the yeast.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antiserum or antibody that recognizes yeast or a yeast fragment as an antigen, a method for preparing these antisera or antibodies, and a method for using these antisera or antibodies.

[0002]

2. Description of the Related Art The technology of gene manipulation that began in the 1970s is used as a genetic engineering for the production of pharmaceuticals such as hormones and vaccines and agricultural crops, etc., and is essential for our daily life. It is permeating as a thing.

Microorganisms play an important role in performing genetic manipulation. There are various types of microorganisms such as Escherichia coli and Bacillus subtilis. Among them, yeast can secrete and produce foreign gene products and can add sugar chains to proteins. Since it has been used as one of the above, it has the merit that the technology such as culture and operation has already been completed.

Actually, for example, hepatitis B vaccine and the like are produced using yeast as a host.

[0005]

2. Description of the Related Art The above-mentioned B prepared using yeast as a host
When a protein such as hepatitis B vaccine is used as a medicine, it is necessary to provide a highly pure product (protein).

[0006] When a protein or the like is produced using yeast as a host, the desired protein is secreted into the culture solution, but it is not easy to purify this protein, and even with the utmost care, yeast is still used. Alternatively, the possibility of contamination by yeast fragments such as cell wall fragments cannot be eliminated. In fact, according to the biological standards of the Ministry of Health and Welfare, it is necessary to confirm the absence of yeast in proteins and the like produced using yeast as a host by performing a culture operation.

The method of culturing yeast for such proteins and the like has a problem that it takes a lot of days to obtain a result, and a special laboratory for culturing is required.

Further, such a method has a problem that only complete contamination with yeast can be detected. For example, when the contaminant is a yeast fragment represented by the yeast cell wall, the yeast does not reproduce even if the culture operation is performed.

[0009]

If the present inventors can obtain an antibody or the like that recognizes yeast or a yeast fragment as an antigen, the present inventors can use these antibodies and the like to solve the problems of these conventional techniques. We thought that it could be solved, and conducted intensive studies. As a result, they have found that by immunizing a vertebrate with yeast or a yeast fragment, an antibody or the like that recognizes this as an antigen can be obtained, and the present invention has been completed.

The present invention is an antiserum or antibody that recognizes yeast or yeast fragments as an antigen, and a method for preparing these antisera or antibodies, which comprises immunizing vertebrates with yeast or yeast fragments.

The present invention also provides a method for detecting contamination with yeast or yeast fragments, which comprises adding these antisera or antibodies to a sample, and further a method for removing or extracting yeast or yeast fragments. Hereinafter, the present invention will be described in detail.

The antiserum or antibody that recognizes yeast or yeast fragment as an antigen is a yeast fragment prepared by physical or chemical treatment of yeast or yeast such as ultrasonic treatment, homogenization, detergent treatment, etc. in vertebrates. It can be prepared by immunization. It should be noted that the yeast referred to here means both the cells isolated from the culture medium and the cells suspended in the culture medium.

As vertebrates immunizing with these antigens,
For example, a rabbit, a mouse, and a goat can be exemplified.
There is no particular limitation.

Yeast or yeast fragments used as an antigen are Pichia genus and Saccharomyces genus (Sacc).
There is no particular limitation as long as it is a yeast or a fragment thereof such as halomyces). Therefore, select the desired yeast,
It can be used as an antigen. Among them, those that recognize yeast or a fragment thereof belonging to the genus Pichia, which is suitable for expressing foreign proteins, as an antigen are useful.

When immunizing vertebrates with yeast or yeast fragments, it is preferable to repeatedly administer the antigen for efficiency. When immunizing a vertebrate, it is preferable to mix an antigen and Freund's adjuvant.

The antiserum of the present invention can be prepared by immunizing a vertebrate with yeast or a yeast fragment as described above, and then preparing it from this vertebrate according to a usual method. The antibody of the present invention can be obtained in an immunoglobulin fraction by subjecting this antiserum to ammonium sulfate fractionation treatment. Furthermore, by applying the obtained immunoglobulin fraction to a column packed with protein A or the like, an immunoglobulin (antibody) belonging to any class can be prepared.

The antibody recognizing the yeast or yeast fragment of the present invention as an antigen also includes a fragmented one by pepsin treatment or the like. Examples of the fragmented antibody include F (ab ′) ₂ fragments (fragments) obtained by treatment with pepsin.

The antiserum or antibody that recognizes the yeast or yeast fragment obtained as above as an antigen naturally has a high affinity for the antigen. Therefore, by using these, it becomes possible to detect whether or not these antigens are present in the sample.

That is, when the antiserum or antibody of the present invention is added to a sample, an immune reaction occurs in the sample with contaminants such as yeast or yeast fragments, resulting in immunoprecipitates. Therefore, when immunoprecipitates are generated, it can be judged that the sample is contaminated with yeast or yeast fragments. The sample is a yeast culture solution (particularly, a culture solution in the case of producing an exogenous protein using yeast as a host), and sometimes a general river, marshes, industrial wastewater, or the like.

In this method, in particular, the antiserum or antibody of the present invention is immobilized on a suitable solid phase, and this is brought into contact with a sample, and the antiserum or antibody of the present invention is a detectable labeling substance. It can be preferably carried out by adding a compound combined with. More specifically, the enzyme or yeast fragment in the sample is captured by the immobilized antiserum or antibody, and the antiserum or antibody bound to the labeling substance is bound thereto, and the antiserum bound to the excess labeling substance is bound. Alternatively, the labeling substance is detected after removing the antibody.

The solid phase is generally an immunoassay method (imm
As long as it is used for unoassay), it may be bead-shaped or plate-shaped. The labeling substance is not limited as long as it can be detected, and is a substance that can be detected by itself, such as a radioisotope, a fluorescent substance, or a luminescent substance, or a substance that causes a detectable change in a substrate, such as an enzyme. May be. In the present invention, it is preferable to use an enzyme as a labeling substance in view of sensitivity and safety.

From the other viewpoint, the above detection method comprises adding the antiserum or antibody of the present invention to a sample,
A method for removing yeast or yeast fragments from a sample. As mentioned above, the addition of the antisera or antibodies of the invention to a sample causes the yeast or yeast fragments in the sample to immunoprecipitate. Therefore, this precipitate may be removed by centrifugation, gel filtration or ultrafiltration membrane. This method also
It can be carried out more efficiently by immobilizing the antiserum or antibody. That is, by immobilizing the antiserum or antibody on a solid phase such as a suitable gel and filling it in a column, contaminants such as yeast or yeast fragments in the sample can be removed simply by applying the sample to the column. , Can process large amounts of sample in a shorter period of time.

Further, the present invention is a method for extracting yeast or yeast fragments in a sample. The operation itself is the same as the method for removing yeast or yeast fragments from the sample described above. This method is useful, for example, for screening new yeasts.

Also in this extraction method, it is preferable that the antiserum or the antibody is immobilized on a solid phase such as a gel and the column is filled with the antiserum.

[0025]

EXAMPLES Examples will be described below to explain the present invention in more detail, but these examples do not limit the present invention.

Example 1 Yeast (Pichia Pastor; Pichia Pastor)
is) to YPD medium (Yeast extract 2
0 g / L, pepton 20 g / L, dextro
(se 20 g / L).

1 ml of this culture solution (8 mg / ml) was added to
The rabbit was injected intravenously using a syringe, and the same operation was repeated every 3 days from the first injection.

Whole blood collection was performed 24 days after the first injection,
This was subjected to centrifugation to obtain serum (10 mg / ml).

The antiserum thus obtained was subjected to ammonium sulfate fractionation to obtain an IgG (immunoglobulin class G) fraction.

This IgG fraction was further treated with pepsin to obtain F (ab ') ₂ fragments.

Example 2 2 ml of a mixture prepared by mixing the Pichia culture solution prepared in Example 1 and complete adjuvant at a ratio of 1: 1 was subcutaneously injected into a rabbit using a syringe. 3 from the first injection
After the day, 2 ml of a mixture obtained by mixing the culture medium and the incomplete adjuvant at a ratio of 1: 1 was subcutaneously injected, and thereafter, the same operation was repeated every 3 days.

Whole blood collection was performed 24 days after the first injection,
This was subjected to centrifugation to obtain serum.

Example 3 The Pichia culture solution prepared in Example 1 was centrifuged to obtain a precipitate, which was crushed with a glass bead and suspended with a phosphate buffer (PBS) to a concentration of 20 mg / ml. It became cloudy.

1 ml of this suspension was intravenously injected into a rabbit using a syringe, and the same operation was repeated every 3 days from the first injection.

Whole blood collection was performed 24 days after the first injection,
This was subjected to centrifugation to obtain serum.

Example 4 The Pichia culture solution prepared in Example 1 was centrifuged to obtain a precipitate, which was crushed with a glass bead and suspended in a phosphate buffer (PBS) at 20 mg / ml. It became cloudy.

2 ml of the mixture of suspension and complete adjuvant in a 1: 1 ratio was injected subcutaneously into the rabbit using a syringe. Three days after the first injection, 2 ml of a mixture obtained by mixing the culture medium and the incomplete adjuvant at a ratio of 1: 1 was subcutaneously injected, and thereafter, the same operation was repeated every three days.

Whole blood was collected 24 days after the first injection,
This was subjected to centrifugation to obtain serum.

Example 5 The Pichia culture solution prepared in Example 1 was diluted to 10 ^-2 to 10 ^-6 times with 20 mM sodium acetate buffer (pH 5) to prepare a 96-well immunoassay preparation. 100 μl was injected into each hole of a gland (manufactured by Nunc) and left at 37 ° C. for 2 hours. The solution in the plate was discarded, 100 μl of a PBS solution containing 0.2 wt% skim milk was injected into each well, and the plate was left at 37 ° C. for 2 hours for blocking treatment. The liquid in the plate was discarded and P containing 0.1% BSA was added.
The serum (antibody) obtained in Example 4 diluted 5000 times with a BS solution was injected into each well in an amount of 100 μl, and left at 4 ° C. overnight.

The liquid in the plate was discarded, and 0.1% Tri
After injecting 100 μl of the PBS solution containing tonX-100 into each well and discarding it, the washing operation was repeated 3 times, and then diluted with PBS solution containing 0.1% BSA 3000 times to Prot.
10 ein A-POX (manufactured by BIORAD) in each hole
0 μl was injected and left at 37 ° C. for 1 hour.

The liquid in the plate was discarded, and 0.1% Tri
After injecting 100 μl of the PBS solution containing tonX-100 into each well and discarding it, the washing operation was repeated 3 times, and then Perox
idase Substrate Kit Color development buffer (H ₂ O ₂ , 2,2-'Azinobis (3-eth
ylbenzothiazoline-6-sulfo
nic acid), made by BIO RAD) 1 for each hole
It was injected by 00 μl each and left at 37 ° C. for 30 minutes.

After 15 minutes, 0.5M oxalic ac
100 μl of id was injected into each hole to stop the reaction, and then the absorbance at 415 nm was measured.

The results are shown in FIG. From FIG. 1, Example 4
When the Pichia culture solution is reacted with the serum (antibody) obtained in
It can be seen that the reaction also increases as the amount of Pichia culture solution (concentration of Pichia antigen) increases.

Example 6 The Pichia culture solution prepared in Example 1 was diluted to 10 ⁻³ to 10 ⁻⁶ times with 20 mM sodium acetate buffer (pH 5) to prepare a 96-well immunoassay preparation. 100 μl was injected into each hole of a gland (manufactured by Nunc) and left at 37 ° C. for 2 hours. The solution in the plate was discarded, a PBS solution containing 0.2 wt% of skim milk was injected into each hole in an amount of 100 μm, and the mixture was left at 37 ° C. for 2 hours to perform a blockinb treatment.

The liquid in the plate was discarded, and 0.1% BSA was added.
Ig of Example 1 diluted 5000 times with PBS solution containing
100 μl of each G fraction was injected into each well and left at 4 ° C. overnight.

The liquid in the plate was discarded, and 0.1% Tri
After injecting 100 μl of the PBS solution containing tonX-100 into each well and discarding it, the washing operation was repeated 3 times, and then diluted with PBS solution containing 0.1% BSA 3000 times to Prot.
10 ein A-POX (manufactured by BIORAD) in each hole
0 μl was injected and left at 37 ° C. for 1 hour.

The liquid in the plate was discarded, and 0.1% Tri
After injecting 100 μl of the PBS solution containing tonX-100 into each well and discarding it, the washing operation was repeated 3 times, and then Perox
idase Substrate Kit Color development buffer (H ₂ O ₂ , 2,2-'Azinobis (3-eth
ylbenzothiazoline-6-sulfo
nic acid), made by BIO RAD) 1 for each hole
It was injected by 00 μl each and left at 37 ° C. for 30 minutes.

After 15 minutes, 0.5M oxalic ac
100 μl of id was injected into each hole to stop the reaction, and then the absorbance at 415 nm was measured.

The results are shown in FIG. From FIG. 2, Example 1
It can be seen that, when the IgG fraction obtained in step 2 was reacted with the Pichia culture medium, the reaction also increased as the amount of Pichia culture medium (concentration of Pichia antigen) increased.

Example 7 The F (ab ′) ₂ fragment obtained in Example 1 was adjusted to a concentration of 1 μg / ml with a sodium carbonate buffer solution (pH 9.5), and a 96-well plate for immunoassay was prepared. 100 μl was injected into each hole (made by Nunc) and left at 37 ° C. for 2 hours. The solution in the plate was discarded, 100 μl of PBS solution containing 0.1 wt% casein was injected into each well, and
It was left at 7 ° C. for 2 hours to be subjected to block ing treatment.

20% of the Pichia culture solution prepared in Example 1 was used.
10 ^-2 times to 1 with mM sodium acetate buffer (pH 5)
It was diluted to 0 ^-6 times, and injected by 100μl to each well. Discard the solution in the plate and use 0.1% Triton X-10.
100 μl of PBS solution containing 0 was poured into each well and discarded, and the washing operation was repeated 3 times. Then, 100 μl of the antiserum of Example 1 diluted 5000 times with PBS solution containing 0.1% casein was added to each well. Each of them was injected and left at 37 ° C. for 2 hours.

The solution in the plate was discarded, and Protei was diluted 3000 times with a PBS solution containing 0.1% casein.
n A-POX (manufactured by BIO RAD) is 100 in each hole.
Each μl was injected and left at 37 ° C. for 1 hour.

The liquid in the plate was discarded, and 0.1% Tri
After injecting 100 μl of the PBS solution containing tonX-100 into each well and discarding it, the washing operation was repeated 3 times, and then Perox
idase Substrate Kit Color development buffer (H ₂ O ₂ , 2,2-'Azinobis (3-eth
ylbenzothiazoline-6-sulfo
nic acid), made by BIO RAD) 1 for each hole
It was injected by 00 μl each and left at 37 ° C. for 30 minutes.

After 15 minutes, 0.5M oxalic ac
100 μl of id was injected into each hole to stop the reaction, and then the absorbance at 415 nm was measured.

The results are shown in FIG. From FIG. 3, F (ab
′) It can be seen that also in the sandwich method using ₂ fragments, the reaction increases with an increase in the amount of Pichia culture solution (concentration of Pichia antigen).

Example 8 An expression vector (pAO804) containing a cDNA encoding rabbit serum albumin (RSA) according to the description in the specification of the patent application (Reference No. 91167) dated July 10, 1991 by the same applicant. ) -Incorporated plasmid (pY
RSA3) was prepared and linearized with BamHI and spheroplast method (Cregg, JM et al., Molec. Cell. Biol. 5,
p3376, 1985) and transformed into Pichia pastoris. The transformed Pichia was cultured in BMMY medium, induced with methanol in the medium, and cultured for 4 days to secrete RSA into the culture medium.

The culture solution was subjected to an ultrafiltration membrane (Toso Co., Ltd., T
S-10) was used for concentration (2.5 mg / ml × 3.4).
l), dialyzed against 20 mM Tris-hydrochloric acid (pH 7), and subjected to ion exchange chromatography (manufactured by Toso Co., Ltd.,
DEAE-TOYOPEARL650), 5% (wt /
vol) Zinc acetate equilibrated chelating column (Pharmacia, Chelating Sepharose Fas)
t Flow), high-performance liquid chromatography (manufactured by Toso Corporation, G3000sw xl), and purified RS
A solution (0.8 mg / ml × 7.5 l) was obtained.

1 μg of the F (ab ′) ₂ fragment obtained in Example 1 was added with sodium carbonate buffer (pH 9.5).
/ Ml, and a 96-well immunoassay plate (nun)
(manufactured by Company c), 100 μl each was injected into each hole and left at 37 ° C. for 2 hours.

The solution in the plate was discarded, 100 μl of a PBS solution containing 0.1 wt% of casein was injected into each well, and the plate was left at 37 ° C. for 2 hours for blocking treatment.

100 μl of the crude purified RSA solution obtained as described above was poured into each well and left at 4 ° C. overnight.

The liquid in the plate was discarded, and 0.1% Trit was added.
After injecting 100 μl of a PBS solution containing onX-100 into each well and discarding it three times, the antiserum of Example 1 diluted 5000 times with a PBS solution containing 0.1% casein was prepared in each well. 100 μl each was injected and left at 37 ° C. for 2 hours.

The liquid in the plate was discarded, and 0.1% Trit was added.
After injecting 100 μl of a PBS solution containing onX-100 into each well and discarding it, the washing operation was performed 3 times, and then Pro was diluted 3000 times with a PBS solution containing 0.1% casein.
100 μl of tein A-POX (manufactured by BIO RAD) was injected into each hole and left at 37 ° C. for 1 hour.

The liquid in the plate was discarded, and 0.1% Tri
After injecting 100 μl of the PBS solution containing tonX-100 into each well and discarding it, the washing operation was repeated 3 times, and then Perox
idase Substrate Kit Color development buffer (H ₂ O ₂ , 2,2-'Azinobis (3-eth
ylbenzothiazoline-6-sulfo
nic acid), made by BIO RAD) 1 for each hole
It was injected by 00 μl each and left at 37 ° C. for 30 minutes.

After 15 minutes, 0.5M oxalic ac
100 μl of id was injected into each hole to stop the reaction, and then the absorbance at 415 nm was measured.

As a result, from the crudely purified RSA solution, the RSA
Yeast-derived contaminants such as yeast or yeast fragments at 0.08 wt% of the protein were detected.

[0066]

INDUSTRIAL APPLICABILITY The present invention provides an antiserum or antibody that recognizes yeast or a yeast fragment as an antigen, and a method for preparing the same. Also, by using these antisera or antibodies, the presence of yeast or yeast fragments in a sample can be easily detected,
It is possible to remove them or acquire them.

Therefore, the present invention can easily and quickly detect whether or not a yeast or yeast fragment which is a contaminant for the solution is present in the yeast culture solution containing the protein of interest, and sometimes this is detected. It is possible to remove and even obtain this.

Immunological substances such as antibodies are known to have high affinity for their antigens. Therefore, these methods provided by the present invention are high-sensitivity detection methods and high-rate removal or acquisition methods.

Furthermore, the method for obtaining yeast or yeast fragments provided by the present invention is also suitable for screening new yeasts.

[Brief description of drawings]

FIG. 1 shows the results of Example 5 of the present invention, which is an enzyme-linked immunosorbent assay (ELIS) using the antiserum of the present invention.
It is a calibration curve when yeast or a yeast fragment is detected by A). In the figure, the vertical axis represents the absorbance at 415 nm, and the horizontal axis represents the antibody dilution rate.

FIG. 2 shows the results of Example 6 of the present invention and is a calibration curve when yeast or yeast fragments are detected by ELISA using the antibody of the present invention. In the figure, the vertical axis is 4
The absorbance at 15 nm is shown, and the horizontal axis shows the antibody dilution rate.

FIG. 3 shows the results of Example 7 of the present invention, and is a calibration curve when yeast or yeast fragments are detected by ELISA using the fragmented antibody of the present invention. In the figure, the vertical axis represents the absorbance at 415 nm, and the horizontal axis represents the antibody dilution rate.

Claims (11)

[Claims] 1. An antiserum which recognizes yeast or a yeast fragment as an antigen. 2. The method for preparing an antiserum according to claim 1, wherein the vertebrate is immunized with yeast or a yeast fragment. 3. An antibody which recognizes yeast or a yeast fragment as an antigen. 4. The antibody according to claim 3, which is F (ab ′) ₂ fragmented. 5. The antibody according to claim 3, which comprises preparing an antiserum which recognizes yeast by immunizing a vertebrate with yeast or a yeast fragment and then purifying the antiserum to obtain an immunoglobulin fraction. Preparation method. 6. A method for detecting contamination with yeast or yeast fragments, which comprises adding an antiserum or antibody that recognizes yeast or yeast fragments as an antigen to a sample. 7. A sample is brought into contact with an immobilized antiserum or antibody that recognizes yeast or a yeast fragment as an antigen, and further,
The method according to claim 6, wherein antiserum or antibody that recognizes yeast or a yeast fragment as an antigen and is bound to a detectable labeling substance is added. 8. A method for removing yeast or yeast fragments from a sample, which comprises adding an antiserum or antibody that recognizes yeast or yeast fragments as an antigen to the sample. 9. An antibody which recognizes immobilized yeast or a yeast fragment as an antigen is used.
The method described in. 10. A method for extracting yeast or yeast fragments from a sample, which comprises adding an antiserum or antibody that recognizes yeast or yeast fragments as an antigen to the sample. 11. The method according to claim 10, which comprises using an immobilized antibody that recognizes yeast or a yeast fragment as an antigen.