DE2004869C2 - Use of an i-RNA obtained in the usual way for antibody formation - Google Patents

Description

waves. In addition, the inventive numerically small injection is that with a Injection-related risk is significantly lower, and allergic side effects are also avoided will. ">

According to the invention, the antiserum can be prepared by dissolving the purified immunoribonucleic acid, for example in a physiological saline solution, and injecting the solution into an animal which is used for the preparation of the antiserum. The effective amount may vary depending on the animal used. It is generally between 50 and 2500 μg per 1 kg of body weight in the form of immunoribonucleic acid. At or immediately after the injection, a small amount of antigen is injected into the animal π. A toxin or a toxoid is generally used as the antigen. The antigen must have the same antigenicity as the antigen used to produce the immunoribonucleic acid. The amount of circulating antibodies produced by a single injection of the antigen alone is at most 1 ½ units / ml, even after 1 month, while the amount, if the antigen is used together with the immunoribonucleic acid, is 30-100 Units / ml is achieved within a very short period of time without repeated injections of a large amount of a toxin or toxoid. According to the invention, therefore, the time is greatly reduced for the production of an antiserum, so that a reproducible production expected u <can. Furthermore, the production of the antiserum is very economical, since the immunoribonucleic acid can be produced from the spleen of animals which are used to produce the antiserum. r>

The following examples illustrate the invention.

example 1

Immunoribonucleic acid is extracted from the spleen of mice that have been sufficiently immunized with a live vaccine from -> »Salmonella enteritidis. The acid is purified by the method given below, whereupon 5 mg of the purified immunoribonucleic acid is dissolved in 0.5 ml of a physiological saline solution. The solution ■ ""> is injected intraperitoneally into mice at the same 10- ⁵ mg of a weakly virulent live vaccine from S. subcutaneously injected into the mice enteritidis. After 5 days, is a highly virulent live vaccine from S. enteritidis intrave- ''»Nös injected into the mice to observe the developed immunity.

Immunribonucleic acid is extracted and after the cleaned using the following method:

2 g of a liver isolated from immunized mice are cut into small pieces. After adding 10 ml of a cooled 0.5% sodium naphthalene-1,5-disulfonate solution (NDS), the tissue is homogenized in an ice bath using a glass homogenizer. The homogenate is mixed with a small amount of a 90% (volume / volume) phenol solution containing 0.1% (weight / volume) 8-hydroxyquinoline (Ph-Hq) with cooling. The water phase separated by centrifugation is added an equal volume of a 10 ^J / nigen (weight / volume) sodium p-aminosalicylate solution containing 2% (weight / volume) sodium dodecyl sulfate (PAS-SDS), whereupon the mixture is again with the Ph-Hq is extracted, which makes up half the volume of the aqueous phase. Washing with the Ph-Hq is carried out more than 4 times, until there are no more visible materials between the water phase and the phenol phase. Twice the volume of cold ethanol is added to approximately 7.5 ml of the water phase obtained in this way. The precipitate formed is dissolved in a small amount of 0.1 M NaCl. This amount contains 2 μg / ml potassium polyvinyl sulfate (PVS). Reprecipitation takes place and repeated washing with ethanol.

All measures are carried out at 4 ° C. After removing the ethanol by centrifugation and evaporation, the dried precipitate formed is dissolved in 3 ml of a cold 0.01 M tris-HCl buffer (pH 7.5) ^{containing 10 "3} M MgCl. This solution is then dissolved 1/30 ml deoxyribonuclease (50 ug / ml) was added, and the mixture at 4 ⁰ C is allowed to stand for a period of 30 minutes. After the addition of 1.5 ml of the PAS SDS and 1.5 ml of a l ° / In the above NDS, ribonucleic acid is extracted with the Ph-Hq in the manner described above and precipitated with ethanol. After washing 5 times with ethanol, the precipitate formed from ribonucleic acid is stored at -20 ° C. until use.

A solution of 2 ml of the thus obtained ribonucleic acid in 1 ml of 0.01 M acetate buffer (pH 5.0) containing 0.1 m NaCl, containing 10- ⁴ m MgCl ₂ and 2 _μ ε / ΓηΙ PVS is above added to 30 ml of a 5 to 20% linear cane sugar gradient made in the same buffer. The tubes are centrifuged in a Hitachi RPS-25 rotor (Type 55-PA, Hitachi Lid., Tokyo, Japan) for a period of 16 hours at 35,000 rpm. After an automatic measurement of the extinction at 250 ΐημ using an ISCO density gradient fractionator. (Model 180 from Instrumentation Specialties Company Inc., USA) fractions are collected. The fractions collected in this way are subjected to negative pressure dialysis through cellophane against a 0.15 M NaCl solution. which contains 2 μg / ml PVS, concentrated and precipitated with ethanol and purified.

The following experimental results are obtained:

100% surviving animals observed during an attack of a highly virulent live vaccine - are according to the inventive method, 5 mg Immunribonukleinsäure and 10 ~ ⁵ mg of a weakly virulent live vaccine in mice injected, then 97 are. 5% of the animals in an attack by the highly virulent live vaccine - 10 ~ ⁵ mg of a weakly virulent live vaccine injected alone for comparison, only 0 to survive.

Example 2

Immunribonucleic acid is made from the spleen of guinea pigs that have been treated with a diphtheria toxoicl have been sufficiently immunized in the same manner as in Example 1. 150 mg the immunoribonucleic acids are injected intraperitoneally into guinea pigs. After 10 days, 5 LF a diphtheria toxoid was injected subcutaneously into the guinea pigs. 5 days after the toxoid injection the level of circulating antibodies in the guinea pigs is titrated.

The following results are emelted:

Are immunoribonucleic acid together with a toxoid according to the method according to the invention injected, then one finds a large amount, namely ΙΟΟμ / mI, of circulating antibodies, while if the toxoid alone is injected as a control, only P / a µ / ml are observed. If the immunoribonucleic acid is injected alone, then the level of circulating antibodies is less than 1 / 10OuVmI.

Example 3

150 μg of immunoribonucleic acid (hereinafter abbreviated to referred to as i-RNA) prepared as in the previous example became guinea pigs injected intraperitoneally. Four weeks later, 5 LF of diphtheria toxoid were injected subcutaneously into the guinea pigs injected. 7 days after the toxoid injection, blood was drawn from the guinea pigs and with Let stand 4 ° C overnight to isolate the serum (antiserum). The serum isolated in this way was Centrifuged for 10 min at 10,000 rpm. The separated, supernatant serum was removed, and the Antibody titers of the antiserum were determined by standard antiserum titrimetry to be the serum antibody level to investigate. The antibody level was determined to be 30 to 100 units / ml.

For comparison and control purposes, the above experiment was repeated, but the Refrain from injecting i-RNA and administer a single dose of the toxoid, either alone or through Substitute the dose of the toxoid for the first dose of i-RNA, followed by the second injection of the toxoid was done, or by omitting the second injection of the toxoid and the first injection of i-RNA alone was administered, etc. The experimental results obtained are shown in the following table.

first dose

Second dose (4 weeks after the first dose of Anlikbodywerl
(Hinhciten / ml) titrated
For the anliscrum.
won 7 days after
the second dose

According to the invention, I-RNS comparison test (1)
No i-RNA injection

Comparative test (2)
Toxoid

Comparison test (3)
i-RNS

Control test (I)
i-RNS

Control test (2)
Toxoid

Toxoid

Toxoid

Toxoid

i-RNS

neither Toxoid nor i-RNS 30-100

0.3-1

30-100

no toxoid injection <0.0l

<0.01

<0.01

When comparing the test results as they 4> were obtained according to the invention, with those of the comparative experiments (1), (3) and the control experiments (1) and (2) show that the injection of the i-RNA before the dose of the toxoid (as an antigen) increases the antibody level of the Antiserum increased significantly compared to cases where the first dose of i-RNA was not administered. If one compares the test result according to the invention with that of the comparative test (2), it can be seen that the two dossn of the toxoid, but without injection of i-RNA, the formation of the antibody to the same extent 5ί as the invention causes, which means that the inventive method in an advantageous manner the Amount of toxoid required to produce the same titer of antibody in the antiserum Able to squeeze half, mi

Example 4

Immunoribonucleic acid was isolated from the spleen of horses which had been sufficiently immunized with the toxoid of a viper, Trimeresurus nukiuanus, and from which the antiserum for the viper oxoid was obtained. The extraction and purification of the i-RNA were carried out in the same manner as in Example 1. 500 μg of the i-RNA were injected intraperitoneally into mice, and 4 weeks later the vipertoxoid was injected into the same mice. The Vipertoxoid used was min by heating at 100 ⁰ C for 10 and pre-contacted with a volume of ethanol. One week after the injection of the viper toxoid, blood was drawn from the mice and the antiserum isolated from the blood. The antibody wri of the isolated serum v / was determined by determining the amount of the antiserum which was necessary to neutralize a given amount of the haemolytic vipertoxoid. The neutralized antibody value (units / m!) Of the antiserum, obtained after the above experiment according to the invention, was found to be 28 units / ml.

For comparison, the above experiment was repeated, but without the initial injection of the i-RNA.

In this comparative experiment (1), the neutralized antibody value was only about 2.3 U / ml. Of the The above experiment according to the invention was repeated again, but the first injection of the i-RNA was caused replaced by a toxoid injection (so there was a first injection of the vipertoxoid, followed by the second vipertoxoid injection 4 weeks later and the

Obtaining the antisemm one week after the / wide injection of the Vipertoxnids). Hey this one Comparative experiment (2) was the neutralized antibody value only 16 U / ml.

When the above test results are compared, it can be seen that the injection of the i-RNA before the Injection of the viper toxoid significantly increased the formation of the antibody compared to the case in which none Injection of i-RNA took place.

U e ι s ρ ι e I>

1 ml of diphtheria toxoid (titer: 40 lf / ml) was subcutaneously administered to rabbits. and 4 weeks later, I ml of the same toxoid was again injected into rabbits / weeks of immunization. i days after the / wide toxoid injection, the rabbits were sacrificed iirici the i-RNA of the sacrificed rabbit was obtained by extracting from the Mil / recovered.

On the other hand, 1 ml of the same diphtheria toxoid was given as the first dose to two adults (as Persons A and H designated) injected subcutaneously, and 4 weeks later again as the second dose the same People A and B (as a comparison test).

I m to demonstrate the inventive effect, I mg of the i-RNA prepared as above was used as the first dose / white adults (as persons ( and D denotes) injected intiapcritoneally, and 4 Weeks later, 1 ml of the diphtheria toxoid was given as a second dose to the same subjects C and D subcutaneously mii / ied.

7 days after injection of the toxoid as a second dose, these subjects received A. B. C and D serum collected and the antibody value per ml of the serum determined by a standard antisenim titrimetry. The results of these tests are summarized in the following table:

: η ·· Γοχοκί 1 ml Toxoid \ η 11 k κ r ρ _ ■ r - Ίοχίΐκΐ 1 ml Toxoid U.n 11-in i-RNS I ml Toxoid li c; t c η / πι 1ι \ : mi i-RNS ! ml Toxoid 1/50 Ii ; mi I / Ml ( i rriL ¹ l / l'i I) ! mi 1/2 (1

Example 6

The procedures of Example 5 were followed using 1 ml of a toxoid from Bacillus Tetanus (Titer: lOLf / ml) instead of the diphtheria toxoid repeated. The results of the test are given in the following table:

: Thu Toxoid : Thu Toxoid Antibody Toxoid Toxoid Weri iF.in- ! -RNS Toxoid hiitcn / mli f- 1 ml ■ -RNS ϊ mi Toxoid 1/80 Q ^: 1 ml I mi 1 / IfX) G i mg i ml 1/10 Il 1 m ° ! ml 1/30

From the above tables it can be seen that the group (People C, D. G and H). the injection of i-RNA plus toxoid produced a significantly higher antibody value than the group (persons A. B. E and

Ί P) who received the two toxoid injections.

The above tests also make it clear that the injection of i-RNA and the injection of Toxoid as an antigen according to the method according to the invention, the unexpected effect of enhancing the i "immunization yields compared to the pail if According to the prior art, two toxoid injections are carried out as antigen.

This results in the further advantage that the inventive use of i-RNS the two times

■ long injection of toxoid or vaecin (as an antigen) a single injection is limited, so that the risk of anaphylactic shock caused by the second injection is limited Injection of toxoid or vaecin. remains minimal. The i-RNA used in the present invention is one of 4 bases compound compound, has no antigenicity and is practically non-toxic, unlike the Toxoids or vaccines. which were previously used for immunization and sometimes anaphylactic Shock and death.

Example 7

a) 2.0 ml of an influenza virus-containing liquid

... the AIIantois cave of the incubated chicken egg to which the Influenza virus had been vaccinated. Rabbits were injected intravenously, and were given 4 weeks later another 2.0 ml of the same fluids containing influenza virus from the allantoic cavity of the chicken egg

i. ben immunized rabbits. One week after the second injection of influenza virus, the rabbits were sacrificed and the immunoribonucleic acid was sacrificed (i-RNA) extracted from the spleen of sacrificed rabbits.

i .. b) 2.0 ml of an antigen (the 2.0 ml of the same Influenza virus-containing fluid from the AIIantois cavity of the chicken egg, as mentioned above, with a additional content of an amount of an oil adjuvant) were given as the first dose to a guinea pig

ι-, injected subcutaneously, and 4 weeks later a another ml of the antigen as before is injected subcutaneously into the same guinea pig as a second dose. 2 Weeks after the second dose of antigen, the serum of the guinea pig treated in this way was

■■ '· melted and the antibody value per ml of serum was after determined using a standard hemagglutination method (Test No. 1). This test was repeated with another guinea pig (Test No. 2).

Further, 1 mg of the i-RNA obtained as above

is given subcutaneously as the first dose to a guinea pig injected, and 4 weeks later 1.0 ml of the the aforementioned antigen (the influenza virus-containing fluid of the Allantois cavity of the chicken egg plus Oil adjuvant) as a second dose of the same guinea pig

- '< chen injected. 2 weeks after the second dose (the injection of the influenza virus-containing liquid), the serum of the thus treated guinea pig was collected and the antibody value per ml of serum by the same standard hemagglutination method as

-5 determined above (Test No. 3). This test was made with repeated with another guinea pig (Test No. 4). The results of these tests are as follows Table summarized:

labile

I -si I DlISIS (Virus). 2 .0 ml No. (Vims). 2 .0 ml I. Anliuen m μ 2 antigen IUU i-RNS. I. I 8 t i-liNS. I. 15 e i s ρ r e

dose

\ nliknr | KT
Wen ι) - / ml)

; ι) a I ormalin-Vak / 'ti of a palhogenic virus, the Japanese l'ncephalitis virus, guinea pig was eh en each in a dose of (V> ml three times with an interval of 1 week / between the virus doses iiuraperitoneal iiiii / ic-rt \ ^:. \ nc Where * n: ii-h ili-r | ptsii_ <n dose of virus, the guinea pigs were sacrificed and the i-RNA that was formed was extracted from the milk / of the sacrificed guinea pigs.

b) 0.5 ml of an antigen (the aforementioned vacc / ins of the Japanese F "ncephalilis-V ^: rus) was injected subcutaneously into a rabbit as the first dose, and 4 weeks later another 0.2 ml of the same antigen (the virus vaccine) was injected subcutaneously. as / wide dose to the rabbit in iii / ier · .. One week after the / wide antigeiuiosis, the scrum ties were treated in this way

Tabel

Antigen (virus). 1.0 ml I / MO

Xnli.L'en (virus), 1.0 ml 1/320

Antigen (virus). 1.0 ml 1/320

\ nliyen (\ ims). 1.0 ml 1/320

Rabbit collected and the antibody level per ml of serum according to a standard complement measurement (Hotle determined (Tesi No. 1). This test was repeated with another rabbit (Test No. 2).

I erncr became 1 mg of the RNA obtained as above injected subcutaneously into a rabbit as the first dose, and 4 VVoi-hi'n cnäli'r wnrrlrn 0? ml ili> v u '<rg'. 'MHP! 1! *.'! < Antigen (of the virus vaccine) is injected into the rabbit as a large dose. One week after the animal dose (animal injection of the virus vac / ins) was the serum from the rabbit treated in this way was collected and the antibody value per ml of serum according to the same Use the Slandard complement fixation method as above. correct (test no.3). This test was repeated with another rabbit (Test No. 4). the Results of these tests are summarized in the following table:

lot I. DnMS (Virus). 0.5 ml No (Virus). 0.5 ml I. antigen mg antigen mg .1 i-RNS., I I 9 4th ! -KNS. I. B e i s ρ i e

\ Dose (V ims). 0.2 ml \ nliknrr.-r- (\ him. 0.2 ml W ₁ teaspoon M / ml) \ ntiuen (\ ι ms ι. 0.2 ml i / SII \ ntiuen l \ iruo. 0.2 ml I / SO Necessary I / IWI \ niii: cn I / SO

a) A paihogenic bacterium. Klebsieila pneumoniae. was incubated for 20 days and then treated with 1% aqueous formalin plus 0.02% aqueous Chrome alum 5 days weak. The cells of the bacterium so weakened were collected and Rabbits are injected intravenously four times with an interval of 1 week between single doses. Seven days after the last dose of the bacterial cells, the rabbits were inoculated and the i-RNA formed extracted from the spleen of sacrificed rabbits.

b) 30 mg of an antigen (cells of the weakened Klebsiella pneumoniae) were used as the first dose Guinea pigs were injected subcutaneously, and three weeks later were given an additional 15 mg of the same antigen as second dose injected subcutaneously into the same guinea pig. Two weeks after the second dose of the The antigen was the serum from the treated in this way

Tabel

Guinea pigs obtained and the antibody * ..rt per ml of serum using a standard hemagglutination method certain! (Test No. I). This test was repeated with another guinea pig (test No. 2).

Further, 1 mg of the i-RNA obtained as above was used as first dose injected subcutaneously into a guinea pig. and three weeks later became 15 mg of the foregoing Antigen (of the bacteria / rush) is injected as a second dose into the same guinea pig. Two weeks after the second dose (the injection of the bacterial cells) the serum from the so treated Guinea pigs obtained and the antibody level per ml of serum by the same standard hemagglutination method as determined above (Test No. 3). This test was carried out on another guinea pig repeated (Test No. 4). The results of these tests are summarized in the following table:

Tc-!

No.

I. Dose

2nd dose

Antibody value (U / ml)

! Antigen

30 mg

2 antigen (bacterial cells)

30 me

Antigen (bacterial indicators i / 800

mg

Antigen (bacterial cells) 1/400

mg

Il

12th

ΊΊμ.'1 / UIIL!

h-.l Si

Dom.

SnlikMiivi-W .rl Il / ml

i-KNS. I ηιμ -1 i-KNS. i mi;

Aiiimcn (Haktcr icti / cllon> 1 / S (Id

I. ^ Mi!

Anliuen (Uiiklcricn / cllcnl 1 / Id (I (I.

Claims (1)

Claim: Use of an i-RNA obtained in the usual way together with a species-specific antigen to induce accelerated or increased antibody formation. The invention relates to the use of an i-RNA obtained in a customary manner together with a species-specific antigen to induce accelerated or increased antibody formation. It is already known that ribonucleic acid (hereinafter referred to as immunoribonucleic acid or i-RNA is called), which is made from macrophages, which is derived from the spleen, lymph nodes and / or peritoneal Exudate from animals exposed to an antigen such as pathogenic bacteria, rickettsiae, virus vaccines or toxin immunized after their injection into humans or nonimmune animals in humans or in their own specific immunity to the antigen that is responsible for the immunization has been used can develop. However, if the immunoribonucleic acid is injected into humans or animals, then the level of antibodies generated in humans or animals are low, especially in the blood serum. It has now been found that upon or immediately after injection of immunoribonucleic acid into humans or animals the production of the antibody can then be increased if a small amount of the r> Antigen that is of the same type as the antigen used to make immunoribonucleic acid (e.g. immune ribonucleic acid for diphtheria and diphtheria toxoid) will. In this case, a high level of -to antibodies can quickly occur in humans or in animals be generated. This fact means that the injection of immunoribonucleic acid into humans or animals Conveys information required to generate the specific antibody. In addition, a preliminary state for the generation of the antibody by injecting the immunoribonucleic acid into created the body. Hence, delivery of the same antigen has a rapid generation of high v » Antibody content. Previously, immunity was only used to prevent or treat human or animal diseases by repeated injections of antigen substances, such as vaccines or toxoids, during " developed over a longer period of time. In contrast, injecting a small amount of the antigen has together with the immunoribonucleic acid, a single injection, soon after the injection a sufficient immunity result, without mi Side effects such as allergies occur. Immunity preferably develops about 2 weeks after injection. In carrying out the previously known methodological \ h to the preparation of antisera circulating antibodies can only by repeatedly injecting an antigen are increased for a long period, while in contrast, in the present invention circulating antibodies can be produced by a simple treatment quickly in large quantities. Furthermore, the immunoribonucleic acid used to carry out this method can be obtained in a very economical manner from the spleen of the animals which were sacrificed to collect the antiserum. Immunoribonucleic acid can be made from macrophages that are found in the spleen, in the lymph nodes or in the peritoneal exudate of immunized animals. All animal species can be used as animals that achieve immunity to antigens. Horses, cattle, pigs and sheep are those most common animals. Immunoribonucleic acid can affect humans or animals of the same or different species be applied. This immunoribonucleic acid is specific against the antigen that is used to produce it has been used. Antigens for the production of! Mmunribonucleic acid are pathogenic bacteria, such as for example whooping cough bacilli, typhus bacilli or Pasteurella pestis, pathogenic spirochetes such as for example Wcil'sche spirochetes, pathogenic viruses, such as rabies viruses, smallpox viruses, influenza viruses or encephalitis viruses, as well as toxoids such as for example diphtheria toxoids, tetanus toxoids or snake venom toxoids. Immunoribonucleic acid can be extracted from immune animals and then purified, namely by known methods for the extraction of ribonucleic acid, for example by the method of Kidson and Kirby (J. Mol. Biol. 7, 312-315, 1966). at Carrying out this method, macrophages are obtained from spleen, lymph nodes or peritoneal exudate ground with a liquid containing a nucleic acid inhibitor, with repeated treatment is carried out with phenol. The immunoribonucleic acid extracted in this way can be used in the manner further purified that it is subjected to the action of a proteolytic enzyme, for example Trypsin to remove contaminated protein, which is followed by column chromatography or a Ultracentrifugation follows. The immunoribonucleic acid purified in this way can be used, for example, in the following manner for a preventive injection can be used. The purified immunoribonucleic acid is used in a physiological Saline solution dissolved. This solution can be administered subcutaneously by injection in an amount of 1–10 mg Adults and in half the amount to be administered to children. At the time of injection or immediately afterwards a small amount of the antigen substance is injected. As an antigen becomes common a vaccine or toxoid is used, the vaccine or toxoid having the same antigenicity must have like the antigen that was used to produce the immunoribonucleic acid. In the event of injecting a vaccine or toxoid alone will only release a small amount of immune antibodies r generated by a single injection, while according to the invention with only one injection of the vaccine or Toxoids, immune antibodies are quickly produced in an amount that is JO- to 100 times that of Metige which is generated in the first-mentioned case. The use according to the invention, in which a sufficient immunity is developed within a short period of time lends itself to generating a Protection during severe epidemics or disease