DE2137630A1 - Toxoid formation from bacterial toxins - by treatment with hexamethylene diisocyanate - Google Patents

Abstract

Bacterial toxins, esp. diphtheria and tetanus, are converted into toxoids, i.e. the toxicity is removed while retaining the antigenic props., by treating with hexamethylene diisocyanate. The process takes only a few hrs., and gives active vaccines.

Description

Process for the toxolization of bacterial toxins the subject of the invention is a process for toxoiding bacterial toxins, especially diphtheria and tetanus toxin.

There are a number of methods for toxoiding bacterial toxins. However, only inactivation with formaldehyde has found wide application. The disadvantage of this procedure is that it takes several weeks. In addition, retoxification was observed especially with diphtheria toxoid.

A method for the toxoidization of bacterial toxins has now been found, which is characterized in that the bacterial toxins with hexamethylene diisocyanate are treated As bacterial toxins come mainly toxins of the diphtheria and Tetanus bacteria in question, which are grown in a known manner. Form thereby they are toxins that can be isolated by separation from the bacteria. Decisive at Toxoidation is the elimination of toxicity while preserving it antigenicity, which in vaccines is the decisive property for immune defense, for the formation of antibodies against the toxins.

Hexamethylene diisocyanate converts the toxin into in a few hours Toxoid around. The antigenicity remains unaffected.

The oxides obtained are good antigens and allow effective vaccines to manufacture.

In the method according to the invention, it is expedient to proceed as follows: that you get to an aqueous solution of the toxin in the neutral pH range between about pH 5 and pH 9 hexamethylene diisocyanate expediently in a water-miscible one but against isocyanate -inert solvents such as acetone or dioxane dissolved at room temperature clogs. Based at a concentration of 0.1-0.5% of the toxoidizing agent to the total volume of the batch, the reaction is usually already after about one Hour completed. The hexamethylene diisocyanate not used for toxoidation turns into hexamethylenediamine and carbonic acid through hydrolysis and can after some time e.g. 1 - 4 hours after the start of the reaction by dialysis e.g. against a neutral isotonic sodium chloride solution can be removed.

The elimination of toxicity can begin in the case of tetanus toxin the mouse, in the case of diphtheria toxin on guinea pigs.

The antigenicity is expediently determined in vitro by means of the ability to bind determined by antibodies from antiserum. It can also be used in vivo for tetanus toxin in antibody binding experiments in mice and in tetanus and diphtheria toxins in active Protective experiment on guinea pigs the protective effect in international units are determined.

The tests required are briefly described below.

In this description Lf = Limes floculationis means that Amount of antigen that flocculates 1 IU of the specific antiseruin; dlm the dose letalis minina, i.e. the smallest dose of diphtheria toxin that weighs 250 g Kills guinea pigs within 4 days after subcutaneous administration.

The protective effect is measured in IU antitoxin / ml serum of the vaccinee.

Antibody binding test in vivo For the detection of the antibody binding capacity of the detoxified tetanus toxin are per 0.4 ml of the following approaches subcutaneously per Mouse injected: 1 ml of the tetanus toxoid prepared in accordance with the invention into the Dilutions of 10, 9, 8, 7, 6, 5, 4, 3, 2 or

1 lf / ml + 1 mol tetanus antitoxin 2 IU / ml - 20 min. At room temperature + 1 ml tetanus toxin # 1.1 Lf / ml - 20 lein. at room temperature + 1 ml NaCl solution (0.9%) With optimal antibody binding, all animals must include the toxoid dilution Kill with 1 Lf / Sl within 4 days.

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

The specific antitoxin is diluted to 100 Lr / ml.

In a given constant volume of antitoxin, starting out of the volume ratio 1: 1, decreasing volumes of the toxin or toxoid added and incubated in a water bath at 5000. The occurrence of the first flocculation reaction observed. This antigen / antibody equivalence point becomes calculated as the Lf value from the volume ratios.

Toxicity and efficacy test for diphtheria toxin 5 guinea pigs (270-320 g) each receive 0.5 ml of the diphtheria toxin adjusted to 50 Lf / ml. Do the animals die or show diphtheria within the next 6 weeks The preparation is considered to be toxic to paralysis.

After the 6th week, the surviving animals are exposed to -10 dlm Diphtheria toxin exposed subcutaneously. If the animals survive the stress, that applies Toxoid as effective. The degree of effectiveness is determined by determining the humoral Antibody titer determined on the 10th day after intoxication.

Toxicity test for tetanus toxin at the @aus die for tetanus toxicity The sample to be tested is obtained in a dilution series with decreasing concentrations Tetanus antitoxin (serum from an immunized horse) for at least 10 minutes Incubated at 200C. In each case, 0.4 ml of the toxin-antitoxin mixtures are used subcutaneously injected into the groin. Two animals do not get treated with sntitoxin Toxin.

If all mice survive up to the 4th day, the preparation is considered not to be toxic.

Example 1: Tetanus toxin 1000 ml of a obtained in a known manner Tetanus toxins with 35,000 Lf / g nitrogen and 5 mg tetanus toxin / ml in 0.1 molar Phosphate buffer pH 7.2 with 2.5 ml of a 10% by volume acetone solution of Hexamethylene diisocyanate is added at room temperature. After an hour it will Reaction mixture placed in a dialysis vessel and 16 hours against neutral isotonic Dialyzed sodium chloride solution.

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

The product of the process resulted in an active protection test on guinea pigs 480 IU / ml.

Example 2: Diphtheria box 1000 ml of a obtained in a known manner Diphtheria toxins with 2500 Lf / mg nitrogen and 3.7 mg diphtheria toxin / ml in 0> 1 molar phosphate buffer pH 7.2 - at room temperature with 5 ml of a 10% acetone solution of hexamethylene diisocyanate added. After an hour it will the reaction mixture placed in a dialysis vessel and 16 hours against neutral dialyzed isotonic sodium chloride solution.

The product of the process is tested for toxicity on guinea pigs and it was found that all toxin by the method of the invention has been converted to toxoid by lexamethylene diisocyanate.

The antigenicity was also tested on guinea pigs, with the antibody titer was 8 IU / ml.

Claims (1)

Claim Process for the toxoidation of bacterial toxins, characterized in that that the bacterial toxins are treated with hexamethylene diisocyanate.