JPH0774161B2 - Toxoidization method of toxin - Google Patents

Description

The present invention relates to a toxoidization method that eliminates toxicity of a toxin produced by a microorganism while retaining its antigenicity, and particularly relates to a toxoidization method in which the obtained toxoid does not cause reversion to toxicity. .

BACKGROUND ART In order to toxoid (detoxify) a toxin produced by a microorganism, a method of mainly treating with formalin and geltaraldehyde has been conventionally used.

It is known that formalin-detoxified toxonoid shows a phenomenon in which toxicity is restored while the toxoid is retained. In order to prevent this reversion of toxicity, for example, a method of adding an amino acid such as L-lysine in the case of diphtheria toxin and glycine in the case of cholera toxin together with formalin to form toxoid has been developed. However, the toxoidization method of adding an amino acid is not always effective for all kinds of toxins produced by microorganisms. For example, in the case of Exotoxin A and P. pertussis toxin of Pseudomonas aeruginosa, it is known that adding an amino acid to form toxoid decreases the antigenicity. On the other hand, the method of preparing toxoid using glutaraldehyde has not been used in the actual industry.

Based on such technical background, the present invention provides a novel toxoidization method in which toxins produced by microorganisms are toxoidized, having sufficient antigenicity, no toxicity, and no reversion to toxicity. The purpose is to do.

As a result of earnest studies, the present inventors have found that the above object can be achieved by treating a toxin in the presence of a specific compound or ion. Thus, according to the present invention, there is provided a toxoidization method characterized by reacting a toxin produced by a microorganism with urea, magnesium ion, or a chaotropic ion whose Hofmeister series is smaller than chloride ion, and formalin. .

Suitable examples of chaotropic ions whose Hoffmeister series are smaller than chlorine ions used in the present invention are thiocyanate ions (SCN ⁻ ), iodine ions (I ⁻ ), and these ions are generally added as sodium salts. To be done. As is well known, a chaotropic ion is a general term for a monovalent anion having a large ionic radius, and the Hofmeister series shows that such an ion dissociates a hydrophobic molecule in a solvent to make it hydrophobic. It corresponds to the effect of weakening the bond. The toxoidization method of the present invention can also be carried out by treating a toxin in the presence of magnesium ions, and magnesium ions are generally preferably added as magnesium chloride. Furthermore, according to the present invention, urea can also provide a toxoid that does not cause reversion to toxicity.

According to the present invention, a toxoid that does not cause reversion of toxicity can be obtained by treating a toxin in the presence of a compound or ion as described above, in addition to the detoxification effect of formalin, those compounds or ions are denatured or dissociated. It acts as an agent, denatures the toxin, dissociates it into subunits, and causes an irreversible change in its three-dimensional structure.
Understood to prevent returning to the original native toxin.

In order to carry out the method of the present invention, it is preferable to add and act on a specific compound or ion as described above to toxin, and then to act on formalin, but at the same time as treatment with formalin or after treatment with formalin, those It is possible to act with the compound or ion.
Specifically, toxin produced by microorganisms is subjected to ammonium sulfate salting out, centrifugation,
Purify or partially purify by an appropriate method such as column chromatography or sucrose gradient centrifugation, and put the purified toxin into a dialysis tube at a concentration of 50 to 500 μg / ml toxin, and act as a denaturant or dissociator. It is dialyzed against a buffer containing the above-mentioned specific compound or ion.
This dialysis contains, for example, 1 to 8 M, preferably 8 M urea, 1 to 5 M, preferably 5 M magnesium chloride, or 0.5 to 3 M, preferably 1 M sodium thiocyanate per 1 volume of the toxin solution. pH 4 ~ 11.0 buffer solution 5 ~ 1000
To Yong. After dialysis, in toxin solution, for example, 0.2
Add formalin to a concentration of ~ 2.0 ^% / v,
The toxin is reacted with formalin at a temperature of 0 ° C for 1 to 10 days. After the formalin treatment, the formalin and the denaturant or dissociator are removed by a method such as dialysis.

According to the present invention, the toxin (protein toxin) produced by the microorganism can be detoxified by the treatment as described above, and a toxoid that does not revert to toxicity can be obtained.

The toxoidization method of the present invention is an industrially useful toxoidization method that can be applied to the toxoidization of most proteinaceous toxins in principle by the simple operation as described above.

Hereinafter, the present invention will be described more specifically by way of examples, but these do not limit the present invention.

Example 1 5 ml of 200 μg / ml of commercially available pertussis toxin (manufactured by Kagaku and Serum Therapy Institute) purified from a culture solution of Bordetella pertussis phase I Tohama strain was sampled to obtain a molecular weight cutoff value of 3,500. It was placed in a dialysis membrane tube and dialyzed against 100 ml of pH 7.2 phosphate buffered saline 0.01M. External dialysis solution (100
ml) was dialyzed for 3 days, changing once a day for a total of 3 times. Next, the above toxin solution was dialyzed at room temperature for 2 days against 50 ml of a 0.01 M pH 7.2 phosphate buffered saline containing 1 M sodium thiocyanate to dissociate and denature the toxin. After dialysis, gelatin was added in an amount of 0.02 w / v% and formalin was added in an amount of 0.6 w / v%, and they were mixed well and incubated at 37 ° C for 5 days in an incubator. The obtained toxoid solution was put into a dialysis membrane tube, and dialyzed using 0.01 M phosphate buffered saline of pH 7.2 as an external solution. For dialysis, 1 volume of dialysis solution is used for 1 dialysis solution
The test was performed in a cold room (4 ° C.) using 50 volumes, and the dialysis external solution was changed once a day for a total of 3 times, and the operation was performed for 3 days.

The thus prepared toxoid is diluted with 0.01 M phosphate buffered saline of pH 7.2 to a protein nitrogen amount of 20 μg / ml or less, and a toxicity test, a titer test and a neutralization test are performed. A test for reversing toxicity was conducted.

Example 2 Containing 1M sodium thiocyanate in Example 1
Instead of the 0.01M phosphate buffered saline at pH 7.2, 0.01M phosphate buffered saline at pH 7.2 containing 8M urea was used. Other operations were performed in the same manner as in Example 1.

Example 3 5 ml of commercially available pertussis toxin 200 μm / ml was collected and placed in a dialysis membrane tube having a molecular weight cutoff value of 3500, and 100 ml of pH 8.
5, dialyzed against 0.01M phosphate buffer. External dialysis solution is 1 a day
Exchanged a total of 3 times. Next, the dialysate was replaced with 50 ml of pH 8.5 0.02 M glycine buffer solution containing 5 M magnesium chloride,
The toxin was dissociated and denatured by dialysis at room temperature for 2 days. The toxin that had been dissociatively denatured was treated with toxoid by dialyzing it at 37 ° C for 5 days using a buffer solution containing 0.6w / v% formalin in 0.02M glycine buffer solution containing 5M magnesium chloride and having a pH of 8.5. Turned into In addition, this dialysis
The test was performed with 50 volumes of external dialysate to 1 volume of internal dialysate. The obtained toxoid solution is 50 times volume pH8.5, 0.02M in a cold room (4 ° C).
It was dialyzed against glycine buffer. The external dialysis solution was exchanged once a day, three times in total, and dialyzed for 3 days. Furthermore, the external dialysate is adjusted to pH 7.
2. Replace with 0.01M phosphate buffered saline, and use dialysis external solution as 1
It was exchanged once a day for a total of 3 times and dialyzed for 3 days.

The thus prepared toxoid is diluted with 0.01 M phosphate buffered saline of pH 7.2 to a protein nitrogen amount of 20 μg / ml or less, and a toxicity test, a titer test and a neutralization test are performed. A test for reversing toxicity was conducted.

Example 4 2 mg of commercially available Pseudomonas aeruginosa exotoxin A (manufactured by Chemistry and Serum Therapy Laboratories) 0.5 mg / ml purified from the culture supernatant of Pseudomonas aeruginosa PA103 strain was used as the method described in Example 3. A toxoid of Pseudomonas aeruginosa toxin was prepared in the same manner as in.

Example 5 Using 2 ml of 0.5 mg / ml of commercially available Pseudomonas aeruginosa exotoxin A (Chemical and Serum Therapy Laboratory), a toxoid of Pseudomonas aeruginosa toxin was prepared in exactly the same manner as in Example 3.

Example 6 0.5 mg / ml of commercially available Pseudomonas aeruginosa exotoxin A (manufactured by Kaketsuken)
A toxoid of Pseudomonas aeruginosa toxin was prepared in exactly the same manner as described in Example 3 using 2 ml.

Example 7 1 mg / ml of a commercially available cholera toxin (Chemical and Serum Therapy Laboratory) purified from the culture solution of Vibrio cholerae (lnaba 569B strain) of Vibrio cholerae was used in the same manner as in Example 1. Cholera toxoid was prepared.

Example 8 1 mg / ml of commercially available cholera toxin (manufactured by Chemistry and Serum Therapy Institute)
Cholera toxoid was prepared in exactly the same manner as described in Example 2 using 1 ml.

Example 9 1 mg / ml of commercially available cholera toxin (manufactured by Chemistry and Serum Therapy Institute)
Cholera toxoid was prepared in exactly the same manner as described in Example 3 using 1 ml.

Table 1 shows the results of the toxicity test, toxicity reversion test, and potency test of the toxoid obtained in the above-mentioned examples. According to the present invention, the pertussis toxoid, the Pseudomonas aeruginosa exotoxin A toxoid and the cholera toxoid prepared by treatment with a modifier or dissociator such as sodium thiocyanate, urea and magnesium chloride and treated with formalin are completely detoxified. However, the phenomenon of reversal of toxicity was not recognized, and the titer was more than the control.

Explanation of Tables 1) Toxoid: Protein 50 μg / ml was used.

2) Toxoid toxicity test: In the case of pertussis toxoid, the mouse leukocyte number increase test is 0.5 Unit / ml or less and the mouse histamine sensitization test is 0.8U in the national test.
Statistical processing was performed for both nit / ml or less, and those that were satisfied were judged to be acceptable. In the toxicity test of the Pseudomonas aeruginosa exotoxin A toxoid, color change using CHO cells was performed, and those that did not cause color change were passed. In the toxicity test of cholera toxoid, those with negative PF activity were accepted.

3) Toxoid reversion test: Toxoid was incubated in a 37 ° C. incubator for 3 weeks, and the toxicity test of 2) was performed.

4) Titer test: The pertussis toxoid titer test was carried out in accordance with the pertussis vaccine titer test method based on the biologics. In the titer test of Pseudomonas aeruginosa exotoxin A toxoid, mice immunized with toxoid were challenged with 100> D ₅₀ of Pseudomonas aeruginosa exotoxin A to examine the survival rate. In the cholera toxoid titer test, an antiserum was prepared by immunizing a rabbit with cholera toxoid, and a cholera toxin neutralization test was performed.

Claims (1)

[Claims] 1. A toxoidization method, which comprises reacting a toxin produced by a microorganism with urea, magnesium ion, or a chaotropic ion whose Hofmeister series is smaller than chlorine ion, and formalin.