DE2137630C3 - Process for the toxoidation of bacterial toxins - Google Patents

Description

Antibody binding test in vivo

To demonstrate the antibody binding capacity of the detoxified tetanus toxin, 0.4 ml of the following approaches are injected subcutaneously per mouse:
2n 1 ml of the tetra prepared according to the invention

liiis toxoids in the dilutions of 10, 9, S, 7, fi, 5.4, λ. 2 h / w. 1 Lf ml
+ I ml tetanus antitoxin 2 IU / ml 20 min. At room temperature
\ - 1 ml tetanus toxin - 1.1 Lf ml -

20 min. At room temperature
+ 1 ml NaCI solution (0.9 ^ Ig)
If the antibody combination is optimal, all animals including the toxoid dilution with 1 Ll, ml must perish within 4 days.

Determination of the Lf value
with diphtheria or tetanus toxin or toxoid.

The specific antitoxin is diluted to 100 Lf / ml. In a given constant volume antitoxin, based on the volume ratio 1: 1, falling volumes of the toxin or toxoid added and incubated in a water bath at 50 "C. In Short disconnection will cause the first flocculation reaction to occur observed. This antigen / antibody equivalents / point is calculated as the Lf value from the volume ratios.

Toxicity and effectiveness test
for diphtheria toxin

5 guinea pigs (270 to 320 g) each receive 0.5 ml of the diphtheric toxin adjusted to 50 Lf / ml. If the animals perish within the following f) weeks or if they show diphthcratic paralysis, so the preparation is considered toxic.

After the 6th week, the surviving animals are exposed to 10 dim diphtheria toxin subcutaneously. If the animals survive the stress, the i'oxoid is considered effective. The measure of effectiveness becomes determined by determining the humoral target animal on the 10th day after intoxication.

Toxicity test for tetanus toxin in the mouse

The sample to be tested for tetanus toxicity is diluted with decreasing concentrations incubated on tetanus antitoxin (serum from an immunized horse) for at least 10 minutes at 20 "C." Two mice each are 0.4 ml of the toxin-antitoxin mixture subcutaneously in the groin injected. Two animals received toxin not treated with antitoxin.

If all mice survive up to the 4th day, the prenate is considered non-toxic.

Example 1: Tetanus Toxoid

H) OU inl one obtained in a known manner Tetanus oxins with 35,000 Lf / mg nitrogen and 5 mg Tetanus toxin / ml in 0.1 molar phosphate buffer pH 7.2 are mixed with 2.5 ml of a 10 percent by volume acetone solution of hexamethylene diisocyanate added at room temperature. After one hour the reaction mixture is placed in a dialysis vessel and against neutral isotonic sodium chloride solution for 16 hours dialysicrt.

The product obtained no longer contained any toxin, as could be demonstrated in the mouse. All toxin was converted into toxoid.

In an active protection test on guinea pigs, the process product gave 480 IU / ml.

Example 2: Diphtheria Toxoid

1000 ml of a diphtheric toxin obtained in a known manner with 2500 Li / ml nitrogen and 3.7 mg diphtheria toxin / ml in 0.1 molar phosphate buffer pH 7.2 are at room temperature with 5 ml of a 10% acetone solution of hex? "" * - ethylene diisocyanate added. After one hour, the reaction mixture is poured into a dialysis vessel and 16 hours against neutral isotonic sodium chloride solution dialyzed.

The product of the process is tested for toxicity on guinea pigs and it was found that everything Toxin converted into toxoid by the process according to the invention by hexamethylene diisocyanate has been.

The antigenicity was also tested on guinea pigs, with an antibody titer of 8 I.Ii.'ml resulted.

Example 3: Gas Fire Toxoid

33 ml of the ultra concentrate of a bacteria-free culture solution of the gas fire pathogen Clostridium novyi with a content of 6600 dlni-ml and one immunological binding capacity of 60 I.U. CI. novyi-antitoxin / ml are with isotonic saline solution made up to a volume of 100 ml and with 0.5 ml of a 10 percent by volume acetone solution of hexamethylene diisocyanate at room temperature offset. After 1 hour, the reaction mixture is counteracted for 16 hours in a dialysis tube dialyzed neutral isotonic saline solution.

To check the detoxification are 10 mice administered subcutaneously per 1 ml of the product obtained. No animal got sick, so everything was toxin converted into toxoid.

The product of the process was 1: 5 with isotonic Diluted saline solution and mixed with 20 percent by volume of a 1% aluminum hydroxide gel.

10 guinea pigs were each with 0.5 ml of this

Suspension immunized once subcutaneously and bled after 21 days. The sera from the animals were pooled.

The examination showed that 1 ml of the serum pool 5 IU Cl. novyi antitoxin.

A preparation detoxified with formaldehyde that was carried under the same conditions led to one Antitoxin content of the scrum pool of 3 IU / ml.

, ₅ Example 4: Cholera Toxoid

47.5 ml of a partially purified cholera toxin in 0.1 molar phosphate buffer pH 7.2 with a proline content of 5 mg / ml, of which 1.2 mg / ml in the haemagglutination inhibition test certain cholera toxin, are used. 10 ml each of this solution are taken with hexamethylene diisocyanate at room temperature Stirring implemented. For this purpose, 5 times at hourly intervals per IO μΙ of a 10% acetone solution of hexamethylene diisocyanate added. One lets

»5 the reaction mixture after 2 hours at room temperature and then stir for 20 hours at + 4 ° C. After that, the solution is in a dialysis tube 16 hours at 4 "C against a buffer following Composition dialysicrt:

0.02 M phosphoric acid

0.5 mM ethylenediaminetetraacetic acid adjusted to pH 8.0 with 2 η sodium hydroxide solution

The reaction product contained as determined in the agglutination inhibition test 0.6 mg / ml immunologically active cholera toxoid.

With the help of the so-called »loop test«, the product of the process is tested on a jejunal loop Dog tested for cholera toxic activity. In the first section of the intestine (loop 1), 2 ml of a 1:24 diluted solution of the untreated cholera toxin, in the second section of the intestine (loop 2) 2 ml one diluted 1:12 with hexamethylene diisocyanate reacted sample injected. 4.5 hours after administration of the two preparations, 107 ml Fluid drained while Loop 2 could not recover fluid.

Thus it is proven that the converted cholera toxin by treatment of hexamethylene diisocyanate lost its toxicity

Claims (1)

Claim: Process for the toxoidization of bacterial toxins, characterized in that the Bacterial toxins are treated with hexamethylene diisocyanate. The invention relates to a method for the toxoidization of bacterial toxins, in particular of diphtheria and tetanus toxin. There are a number of methods for toxoiding bacterial toxins. However, it has wide application only found inactivation with formaldehyde. This method has the disadvantage that there are several Takes weeks. In addition, especially with diphtheria toxoid. a retoxification. A method for the toxoidization of bacterial toxins has now been found, which is characterized is that the bacterial toxins are treated with hexamethylene diisocyanate. The main bacterial toxins that come into question are toxins from diphtheria and tetanus bacteria be bred in a known manner. In doing so, they form toxins by separating them from the bacteria can be isolated. In toxoidation, the elimination of toxicity while preserving it is crucial the antigenicity, which in vaccines is the decisive property for the immune defense, for the Representing the formation of antibodies against the toxins. Hexamethylene diisocyanate converts the toxin into a toxoid in a few hours. It remains the antigenicity unaffected. The toxoids obtained are good antigens and allow effective vaccines to manufacture. The process according to the invention is advantageously carried out by adding hexamethylene diisocyanate to an aqueous solution of the toxin in the neutral pH range between about pH 5 and lppH 9, expediently in a water-miscible but isocyanate-inert solvent such as acetone or dioxane dissolved at room temperature. At a concentration of 0.1 to 0.5 % of the toxoidizing agent, based on the total volume of the batch, the reaction is usually complete after about an hour. The hexamethylene diisocyanate not consumed for toxoidation is transformed by hydrolysis into hexamethylene diamine and carbonic acid. B. 1 to 4 hours after the start of the reaction can be removed by dialysis, for example against a neutral isotonic sodium chloride solution. The elimination of toxicity can be done in the case of the tetanus toxin in the mouse, in the case of the diphtheria toxin be checked on guinea pigs. The antigenicity is expediently determined in vitro by means of the binding capacity of antibodies from antiserum certainly. In addition, in the case of the Tetaiius toxin, an antibody binding test can be carried out in vivo on the Mouse and tetanus and Dinhtheria toxin in active protection experiments on guinea pigs Protective effect can be determined in international units. The tests required are briefly described below. In this description: Lf = Limes Noculationis, the amount of antigen the 1 1.1Ϊ. of the specific Aii! "~ rum uusf lures: dim the letalis minima dose, d. i. the smallest ίο dose of diphtheria toxin. the one heavy 25Og After subcutaneous administration, guinea pigs are killed within 4 days.
The protective effect is measured in IU anlitoxin / ml serum of the vaccinee.