JPS63166835A - Conversion of toxin to toxoid - Google Patents

Abstract

PURPOSE:To convert toxin produced by microorganism to toxoid and prevent the reversion of toxicity, by treating the toxin with urea, a specific ion and formalin. CONSTITUTION:Toxin produced by microorganism is converted to toxoid by treating the toxin with urea, magnesium ion or a chaotropic ion having lower order in Hofmeister series than chlorine ion (e.g. thiocyanate ion, iodine ion, etc.) and formalin. The chaotropic ion is a general name of univalent anions having large ionic radius and the Hofmeister series corresponds to the effect of such as ion to dissociate a hydrophobic molecule and loosen a hydrophobic bond in a solvent. A toxin having sufficiently high antigenicity and absolutely no toxicity can be produced by this process. The process can be applied to almost all proteinous toxins by simple procedure and is useful from industrial point of view.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a toxoidization method that eliminates toxicity while retaining antigenicity of toxins produced by microorganisms. Regarding the law.

Part I In order to toxoidize (detoxify) toxins produced by microorganisms, a method of treatment with formalin and geltaraldehyde has conventionally been used.

It is known that toxoids that have been detoxified with formalin exhibit a phenomenon in which toxicity returns to those in which the toxoids are stored. To prevent this toxic reversion, e.g.
A method has been developed in which amino acids such as L-lysine for diphtheria toxin and glycine for cholera toxin are added together with formalin to form toxoids. However, the toxoidization method by adding amino acids is not necessarily effective against all types of toxins produced by microorganisms. For example, Green II! ! ! In the case of iI exotoxin A and pertussis toxin, it is known that the antigenicity decreases when amino acids are added to make toxoids.

On the other hand, the method of preparing toxoids using geltaraldehyde is not used in actual industry.

Based on this technical background, the present invention is aimed at converting toxoids produced by microorganisms into novel toxoids that have sufficient antigenicity, have no toxicity, and do not cause reversion to toxicity.・The purpose is to provide a method of conversion.

As a result of extensive research, the present inventors have discovered that the above object can be achieved by treating toxins in the presence of specific compounds or ions. Thus, according to the present invention, there is provided a toxoidization method characterized in that urea, magnesium ions, or chaotropic ions in the Hofmeister series smaller than chloride ions, and formalin act on toxins produced by microorganisms. .

Suitable examples of chaotropic ions used in the present invention whose Hofmeister series is smaller than chlorine ions are thiocyanate ions (SCN-) and iodine ions (■-), and these ions are generally added as sodium salts. be done. As is well known, chaotropic ions are a general term for monovalent anions with a large ionic radius, and in the Hofmeister series, such ions dissociate hydrophobic molecules in a solvent and form hydrophobic molecules. This corresponds to the effect of weakening the bond. The toxoidation method of the present invention can also be carried out by treating the toxin in the presence of magnesium ions, which are generally preferably added as 1 cum of magnesium chloride. Furthermore, according to the present invention, a toxoid that does not revert to toxicity even when exposed to urea can be obtained.

By treating toxins in the presence of the above-mentioned compounds or ions according to the present invention, toxoids that do not revert to toxicity can be obtained because, in addition to the attenuating effect of formalin, these compounds or ions can be used as denaturants or dissociators. Acts as an agent, denatures the toxin or dissociates it into subunits, causing irreversible changes in its tertiary structure.
It is understood that this is to prevent a return to the original native toxin.

In order to carry out the method of the present invention, it is preferable to add specific compounds or ions such as those mentioned above to the toxin, and then to react with formalin. It is also possible to act with compounds or ions.

Specifically, toxins produced by microorganisms are removed by salting out ammonium sulfate, centrifugation,
Purification or partial purification is performed by an appropriate method such as column chromatography or sucrose gradient centrifugation, and the purified toxin is placed in a dialysis tube at a concentration of, for example, 50 to 500 μg/ml of toxin, and acts as a denaturing or dissociating agent. Dialyze against a wt city fluid containing the above-mentioned specific compounds or ions. This dialysis is performed against toxin solution, volume of fluid, e.g.
1-8M, preferably 8M urea, 1-5M, preferably 5M magnesium chloride, or 0.5-3M,
After dialysis against 5-1000 volumes of 1) H4-11.0 buffer solution, preferably containing IM sodium thiocyanate, for example, 0.2-2.
Add formalin to a concentration of 0'/v,
The toxin and formalin are reacted for 1 to 10 days at a temperature of 0°C. After formalin treatment, formalin and the denaturing agent or dissociating agent are removed by a method such as dialysis.

According to the present invention, toxins produced by microorganisms (<N white toxin) can be detoxified by the above-described treatment, and toxoids that do not revert to toxicity can be obtained.

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

EXAMPLES The present invention will be explained in more detail with reference to Examples below, but these are not intended to limit the present invention.

Commercially available pertussis toxin (Chemo and Serum Therapy Research Institute %1) 200 μ3 /
511 ml were distilled, and the molecular weight cutoff value was 3,
Pour into a 500 dialysis membrane tube and adjust the pH of 1001 to 7.2.
Phosphate buffered saline 0. Dialyzed into OIM. The dialysis solution (100 ml) was exchanged once a day for a total of 3 times and dialyzed for 3 days. Next, 0.0 with 1M sodium thiocyanate
The toxin solution was dialyzed against buffer 501 of 1M, pH 7, diphosphate buffered saline at room temperature for 2 days to dissociate and denature the toxin. After dialysis, gelatin was added to Q, 02 w/v%,
Formalin was added to Q and 6 w/v%, mixed well, and incubated at 37° C. for 5 days in a thermostat. The obtained toxoid solution was placed in a dialysis membrane tube and dialyzed against a pH 7.20 OIM phosphate buffered saline solution.

Dialysis was performed in a cold room (4° C.) using 50 volumes of the external dialysis solution per 1 volume of the internal dialysis solution, and the external dialysis solution was exchanged with ICI three times a day for 3 days.

The toxoid thus prepared was prepared at pH 7°2.
．． OIM phosphate buffered saline contains 20 μg of protein nitrogen.
The solution was diluted to a concentration of less than /ml, and a toxicity test, a potency test, and a neutralization test were conducted, and a test for toxicity recovery was also conducted.

In place of the 0.01M phosphoric acid*i physiological saline at pH 7.2 containing 1M sodium thiocyanate in Example 1, 0.01M phosphate buffered saline at pH 7.2 containing 8M urea was used. was used. Other operations were performed in the same manner as in Example 1.

Mitatetan J 200 μg/g+l of commercially available pertussis toxin IIj was collected at 5-1, placed in a dialysis membrane tube with a molecular weight cut-off value of 3500, and dialyzed against 1001 pH 8,5,0,01M phosphate buffer.

The external dialysis solution was exchanged once a day, three times in total. Next, add 5M magnesium chloride to the external dialysis solution pllB, 50, 02
The solution was exchanged with M glycine buffer solution 501, and dialysis was performed at room temperature for 2 days to dissociate and denature the toxin. Furthermore, the toxin that has been dissociated and denatured has a pH of 8.5 using 5M magnesium chloride.
Toxoidization was performed by dialyzing at 37° C. for 5 days using a buffer solution prepared by adding 0.6 W/V% formalin to a 0.02 M glycine buffer solution as an external solution. In addition, this dialysis is a dialysis fluid! The test was carried out using 50 volumes of external dialysis solution per volume.

1 lfl of toxoid prepared by 1 inch is stored in a cold room (4℃) at 50
It was dialyzed against Baeya's pf18.5.0.02M glycine buffer. The external dialysis solution was exchanged once a day, a total of 3 times, and dialysis was performed for 3 days. Furthermore, the external dialysis solution was exchanged to pH 7, 2, 0, OIM phosphate buffered saline) α, and the external dialysis solution was added once a day.
The tube was replaced a total of three times and dialysis was performed for three days.

The toxoid thus prepared was prepared at pH 7°2.
．． Protein nitrogen content in OIM phosphate buffered saline 20118
The mixture was diluted to a concentration of /s+I or less, and a toxicity test, a potency test, and a neutralization test were conducted, as well as a test to investigate the reversion of toxicity.

叉"L trouble".

Green HM (Pseudow+onas uyu correction criminal)
Commercially available M. aeruginosa exotoxin A purified from the culture supernatant of strain PA103 (manufactured by Chemo-Serum Therapy Research Institute) 0.5-
A toxoid of Pseudomonas aeruginosa toxin was prepared in exactly the same manner as described in Example 3 using 8/■1 and 2-1.

X Madan J A toxoid of Bacterium aeruginosa toxoid was prepared in exactly the same manner as described in Example 3 using 0.5 mg/ml of commercially available Pseudomonas aeruginosa exotoxin A (manufactured by Chemo-Serotherapy Kenkyushin). .

Friend 1■1 Commercially available green i! ! The toxoid of Pseudomonas aeruginosa toxin was purified using bacterial exotoxin A (Kaketsuken 81) 0.5118/ml in exactly the same manner as described in Example 3. L, ta.

1) Vibrio cholerae (ln
Cholera toxoid was prepared in exactly the same manner as described in Example 1 using commercially available cholera toxin (Chemo and Serum Therapy Research Institute, Japan, 118/1) purified from the culture solution of Aba 5698 strain.

1 weighing commercially available cholera toxin (chemo and serum therapy research new product) 118/
Cholera toxoid was produced by FA in exactly the same manner as described in Example 2 using 11 ml.

1m Commercially available cholera toxin (manufactured by the Institute of Science and Serum Therapy) IB/
Cholera toxoid was prepared in exactly the same manner as described in Example 3 using 11 ml.

The results of the toxicity test, toxicity reversion test, and potency test of the toxoid obtained in the above-mentioned Examples are shown in Table 1. , pertussis toxoid prepared by formalin treatment, Green 1119
Exotoxin I\toxoid and cholera toxoid were both completely detoxified, and the phenomenon of return of toxicity was also observed, and the titer was also higher than the control.

Table description 1) Toxoid: Performed with 50 μg #+l of protein.

2) Toxicity test of toxoid 2 In the case of pertussis toxoid, the mouse white blood cell count increase test specified by the national certification is 0.5 Unit/ml or less and mouse histamine sensitization! 119 is 0.8 Unit/11 or less, and those that satisfy both are judged to be passed.For the toxicity test of Pseudomonas aeruginosa exotoxin A toxoid, a color change is performed using C11θ cells, and a color change is performed. Those that did not cause the problem were considered to have passed. In the toxicity test of cholera toxoid, those with negative PF activity were considered to have passed.

3) Toxoid toxicity reversion test: Toxoid at 37℃
The cells were incubated in a thermostatic chamber for 3 weeks, and the toxicity test described in 2) above was conducted.

4) Potency test 2 The potency test of pertussis toxoid was conducted in accordance with the pertussis vaccine potency test method based on biological product standards. Potency testing of Green IImM exotoxin A toxoid showed 100>D in mice immunized with the toxoid.
se was challenged with Pseudomonas aeruginosa exotoxin A, and the survival rate was examined. For the potency test of cholera toxoid, an antiserum was prepared by immunizing a rabbit with cholera toxoid, and a neutralization test of PF activity by cholera toxin was conducted.

Claims (1)

[Claims] Toxins produced by microorganisms include urea, magnesium ions,
Alternatively, a toxoidization method characterized by using a chaotropic ion whose Hofmeister series is smaller than a chlorine ion, and formalin.