Administration of serum according to Bezredko: algorithm, scheme, for diphtheria, tetanus, butulism

What do PSCHI and PSS vaccines contain?

PSCHI is a vaccine containing specific antitetanus antibodies (100 IU of active substance is contained in 1 ml). Additional components of the drug are represented by glycine, which acts as a stabilizer, water for injection and sodium chloride.

PSS (tetanus serum) is a protein fraction of horse blood serum that has been immunized with tetanus toxoid or toxin. It contains immunoglobulins. Also contains chloroform. The serum is available in the form of a clear or yellow-tinged liquid.

PRODUCTION

The production of antitetanus serum must be validated in order to confirm the established requirements that guarantee its quality and safety of use.

The serum is obtained from the blood plasma of horses hyperimmunized with tetanus toxoid/toxin. To obtain a purified concentrated immunoglobulin fraction of horse blood plasma containing antibodies (antitoxins) that neutralize tetanus exotoxin, methods of salt fractionation, enzymolysis, and membrane filtration are used.

Mechanism of action

Tetanus is one of the most dangerous infectious diseases - the mortality rate for children is about 90%; for adults, this figure ranges from 20 to 80%. Therefore, the easiest way to protect a person from the disease is vaccination. The vaccine contains weakened tetanus (if the vaccine is complex, then diphtheria) toxins.

These toxins retain their immunogenic properties, so once introduced into the body, they trigger an immune response to their presence in the form of the production of specific antibodies. The body remembers the structure of this antigen and, in case of illness, the produced antibodies recognize the pathogen and help actively fight it. Thus, the body develops acquired immunity.

It, in turn, is divided into natural and artificial acquired types. With natural immunity, antibodies are synthesized by the body independently after suffering from illness. The body acquires passive immunity through the introduction of antigens against various diseases artificially - that is, vaccination.

After the vaccine is administered, the maximum concentration of antibodies in the blood is achieved within 1-2 days. The elimination period from the body ranges from 6 to 8 weeks.

pharmachologic effect

The drug Tetanus immunoglobulin is a solution of a protein fraction that is immunologically active, which is isolated from donor plasma using the ethyl alcohol fractionation method at a temperature below 0 °C. Donors were immunized with tetanus toxoid , tested for the absence of antibodies to HIV and hepatitis C, as well as hepatitis B virus antigen. The drug has the property of neutralizing tetanus toxin and provides the creation of immune protection against tetanus.

Indications for vaccination

The drug is used in 2 main cases:

• For the prevention of disease (including emergency).
• For the treatment of tetanus/diphtheria.

Emergency prevention is carried out in case of various injuries (industrial, domestic injuries, frostbite, insect bites, penetrating injuries, splinters, bedsores, etc.), when the causative agent of infection (tetanus bacillus) can enter the body through an open wound.

The main habitat of this microorganism is the soil, where it can remain active for several years. Routine vaccination is carried out according to the established vaccination calendar.

Timing of vaccine administration

The administration of serum according to Bezredko to persons who have been injured must be carried out as soon as possible - within 20 days (if patients were vaccinated against tetanus more than 5 years ago). It is during this period, if the pathogen enters the body, the disease can occur in a latent form - without the manifestation of corresponding symptoms.

If a person has not previously been vaccinated against tetanus/diphtheria or he does not have a full course of scheduled vaccinations, in addition to the vaccine, he is given a special serum. It helps in a short period to create and develop the body specific immunity to the pathogen.

How is vaccination tolerated?

Vaccination against tetanus and diphtheria is usually easily tolerated - without a significant deterioration in well-being. Often vaccination is done with a complex preparation that contains several antibodies at once. For example, vaccination against whooping cough, diphtheria and tetanus (DPT) or tetanus and diphtheria (DT).

This vaccine is more likely to cause adverse reactions because it is multicomponent. To avoid deterioration of health after vaccination, it is recommended to do so within 30 minutes. stay in the hospital - if unpleasant symptoms develop, doctors will provide the necessary assistance.

Possible reactions and complications

The body's reaction to the introduction of a vaccine can be of 2 types:

• Post-vaccination reaction (develops immediately or within a few hours after the vaccine is administered and goes away on its own approximately 3 days after vaccination).
• Post-vaccination complication (causes changes in the body and deterioration in health, which are long-term; therapy may be required).

Types of reactions and possible complications to vaccination are presented in the table below:

When vaccinated in childhood, children may complain of pain while walking, itching at the injection site. In adulthood, the incidence of reactions to vaccination decreases.

It is worth noting that the risk of possible complications after vaccination is hundreds, and in some cases, thousands of times less than the risk of developing complications after an infectious disease.

Compatibility with alcohol and drugs

After administration of tetanus serum, it is prohibited to drink alcoholic beverages for three days.

Otherwise, adverse reactions may develop:

• temperature increase;
• allergic skin rashes;
• intestinal dysbiosis and diarrhea;
• exacerbation of chronic diseases.

It should also be taken into account that the combined administration of serum and tetanus toxoid leads to suppression of the immune response.

Instructions for use

Immunoglobulin serum is administered by intramuscular injection into the anterior lateral thigh of children under 3 years of age. For older children and adults, the vaccine is administered under the shoulder blade or shoulder muscle. Before the injection, the bottle with the drug must be kept for 2 hours at a temperature of 18 to 22 0C.

Opening the bottle and the immediate vaccination procedure is carried out in compliance with the relevant sanitary standards and rules. To avoid the formation of foam in the drug, it is necessary to take a needle with a wide bore to collect the vaccine, and use a different needle to administer the drug.

When treating tetanus, the drug is administered intravenously or into the spinal canal. The dosage of the medicine is determined by the doctor depending on the severity of the disease.

Human tetanus immunoglobulin

Instructions for use:
HUMAN IMMUNOGLOBULIN ANTI-TETANUS,
solution for intramuscular administration 250 IU

Registration number:

LSR-000791/08 dated 02/15/2008

Human tetanus immunoglobulin, solution for intramuscular administration, is a concentrated solution of a purified fraction of immunoglobulins isolated by ethyl alcohol fractionation from the blood plasma of donors immunized with tetanus toxoid and individually tested for the absence of hepatitis B virus surface antigen (HBsAg) and antibodies to hepatitis C virus and viruses human immunodeficiency HIV-1 and HIV-2.

The protein concentration in the preparation is from 10 to 16%. The content of antibodies to tetanus toxin is not less than 50 international units (IU) in 1 ml and not less than 250 IU in 1 ampoule (dose) of the drug. The drug does not contain preservatives or antibiotics. Stabilizer - glycine at a concentration of (2.25±0.75)%. Transparent or slightly opalescent liquid, colorless or slightly yellow. During storage, a slight sediment may appear, which disappears after gentle shaking at a temperature of 18 to 22 °C.

Immunobiological properties.

The active principle of the drug is class G immunoglobulins, which have the activity of antibodies that neutralize tetanus toxin. The maximum concentration of antibodies in the blood is achieved 24-48 hours after administration; The half-life of antibodies from the body is 3-4 weeks.

Purpose.

Emergency prevention of tetanus in adults and children who have not received a full course of immunization with tetanus toxoid or with an unknown vaccination history.

Directions for use and dosage.

The drug is administered intramuscularly once into the upper outer quadrant of the gluteal muscle or into the outer surface of the thigh in a dose of at least 250 IU, regardless of age. The drug should not be administered intravenously.

Before injection, the ampoule with the drug is kept for 2 hours at a temperature of 18 to 22 °C. The opening of the ampoules and the administration procedure are carried out in strict compliance with the rules of asepsis and antiseptics. To avoid the formation of foam, the drug is drawn into a syringe with a wide-bore needle, and another needle is used for injection. The drug cannot be stored in an opened ampoule. The drug is not suitable for use in ampoules with damaged integrity or labeling, if the physical properties have changed (change in color, clouding of the solution, the presence of flakes that do not break), if the expiration date has expired, or if stored improperly.

Side effects.

As a rule, there are no reactions to the administration of immunoglobulin. In rare cases, local reactions may develop in the form of slight hyperemia and pain in the injection area. The temperature may rise to 37.5 °C during the first day after administration of the drug. Individuals with altered reactivity may develop allergic reactions and, in extremely rare cases, anaphylactic shock. In this regard, persons who received the drug must be under medical supervision for at least 30 minutes. Treatment rooms where immunoglobulin is administered must be provided with anti-shock therapy.

Contraindications for use.

The administration of immunoglobulin is contraindicated for persons with a history of severe allergic reactions to the administration of human blood products. For persons suffering from allergic diseases or with a history of clinically significant allergic reactions, antihistamines are recommended on the day of immunoglobulin administration and for the next 3 days. For persons suffering from systemic immunopathological diseases (diseases of the blood, connective tissue, etc.), immunoglobulin should be administered against the background of appropriate therapy.

Data on the administration of immunoglobulin are recorded in the appropriate accounting forms, indicating the batch number, date of manufacture, expiration date, manufacturer, dose and date of administration, nature of the reaction to the administration.

Interaction with other drugs.

The drug can be used in combination with other drugs, provided it is administered as a separate injection, without mixing with other drugs. After administration of immunoglobulin, active immunization against measles, mumps, rubella and tetanus is carried out no earlier than 3 months later.

Release form.

In ampoules, 1 dose (at least 250 IU) in a volume of no more than 5 ml (depending on the specific activity). 10 ampoules in a pack with instructions for use and an ampoule knife or scarifier.

Vacation conditions.

Dispensed with a doctor's prescription.

Best before date. storage and transportation conditions.

Shelf life: 2 years. A drug that has expired cannot be used. Storage and transportation conditions in accordance with SP 3.3.2.1248-03 at a temperature of 2 to 8 °C out of the reach of children. Freezing is not allowed.

Producer.

FSUE NPO Microgen, Russia,
https://www.microgen.ru

Technique for introducing serum using the Bezredko method

Frequently, a French microbiologist who proposed a method of administering therapeutic serums based on reducing or preventing the body’s negative reaction and the development of complications to them (in particular, in the form of anaphylactic shock). This method involves fractional administration of drugs.

This makes it possible to neutralize antibodies and reduce the concentration of active substances in the blood, as a result of which subsequent administration of antigens does not cause the development of complications.

The introduction of serum according to Bezredko has a specific algorithm:

1. The ampoule with the drug must be carefully examined. If the liquid contains sediment, cloudiness, foreign impurities, the ampoule is damaged, or the label with the necessary information is missing, the use of such medication is prohibited.
2. Before opening, the end of the ampoule must be treated with sterile cotton wool. After opening, the ampoule should be covered with a sterile napkin.

   
   Algorithm for serum administration according to Bezredko

3. Before administering a therapeutic dose, it is necessary to test the body's sensitivity to the drug through a test. For this, a special diluted serum is used (comes with the drug and is marked in red). The sample is injected subcutaneously into the arm above the wrist, having previously disinfected the future injection site. The drug is administered with a special tuberculin syringe with a thin needle. As soon as the cut of the needle enters the skin, 0.1 ml of diluted serum is injected. If after 20-30 minutes. the diameter of the papule formed as a result of the injection is less than 0.9 cm, and there is slight redness around it; the test is considered negative.
4. After this, 0.1 ml of undiluted serum should be injected subcutaneously into the outer surface of the shoulder.
5. After half an hour from the moment of injection (if there is no reaction to repeated administration of the drug), the remaining prescribed dose is administered intramuscularly.

The Bezredko method underlies the introduction of most serums used to treat and prevent the development of various diseases.

Antitetanus serum

The serum is administered according to the above algorithm. The dosage of the drug during treatment is 1500-2000 IU for each kg of a person’s weight. Such a high dose allows the tetanotoxin to quickly bind, which, after infection of the body, spreads into the lymph, blood and cerebrospinal fluid of a person, provoking muscle spasms.

If a severe course of the disease is observed, the following serum administration scheme is allowed:

• Intramuscularly - 100 thousand IU is injected (injected into the gluteal muscle or under the shoulder blade).
• Intravenously - up to 50,000 IU. In this case, the indicated dosage is diluted with an isotonic solution of sodium chloride (the ratio is from 1:5 to 1:10) and heated to a temperature of 37 0C.

When the indicated doses are administered, antitoxic antibodies are produced in the body over a period of 3 weeks, so repeated administration of the serum is not required. Additionally, the patient is prescribed a course of antibiotics. If necessary, anti-shock measures can be taken - warming, the use of stimulants of the cardiovascular and nervous systems, blood transfusions, etc.

If the test for sensitivity to anti-tetanus serum shows a positive result, then its administration can be carried out only if there are clear indications (for example, extensive contamination of the wound).

For botulism

The administration of serum according to Bezredko for botulism is carried out according to the general scheme with a mandatory test for sensitivity to the drug. The injection is prescribed immediately after the clinical diagnosis is established, since it has the maximum therapeutic effect in the first 72 hours from the onset of the disease.

The dosage of a drug containing horse antitoxin depends on the type of disease the person has. For example, with serotype B, 15-20 thousand IU must be administered.

If the type is unknown, then a complex of single-component drugs or polyvalent serum is administered according to the following scheme:

• 10,000 IU antitoxin type A;
• 10,000 IU type E antitoxin;
• 15-20 thousand IU of type B antitoxin.

Regardless of the severity of the disease, 1 therapeutic dose of serum must be administered intravenously. It is pre-diluted in 200 ml of warmed saline solution. If the botulism toxin has already penetrated the muscle-nervous junction, the injected antitoxin cannot neutralize it, but it can slow down or completely stop the progression of the disease.

And with wound botulism (the entry of C. Botulinum spores, which cause botulism, into an open wound with their subsequent reproduction) it reduces the risk of complications, including death. To avoid the development of allergic and other undesirable reactions of the body to the administration of serum, the patient should first be injected with 60-90 ml of prednisolone.

Antidiphtheria serum

Diphtheria is a dangerous infectious disease caused by the diphtheria bacillus. In 95%, the infection affects the mucous membranes of the oropharynx. The disease is transmitted through airborne droplets or through direct contact with an infected person. In some cases, cats, horses and dogs can be carriers of the infection.

Anti-diphtheria serum is used to treat diphtheria. Once introduced into the body, it neutralizes the infection toxin that circulates in the blood.

People who are hypersensitive to horse protein in whey may experience anaphylactic shock. To avoid this, before starting therapy, an intradermal test is carried out according to the above-mentioned Bezredko method.

If there is no specially diluted serum, it is prepared independently by mixing 0.1 ml of therapeutic serum with 9.9 ml of sterile sodium chloride solution. If there is a negative reaction to the vaccine, 0.1 ml of the drug should be administered subcutaneously, undiluted, and after 30 minutes. - remaining dose.

The dosage of serum depends on the severity and form of the disease:

• Localized form (pharynx, nose, eye skin, genitals) - 10,000-20,000 IU is administered via intramuscular injection.
• Oropharynx with distribution (plaque appears on several organs, for example, on the tonsils and larynx) - 20,000-30,000 IU is also administered intramuscularly.

For toxic form of diphtheria

After a week after the disease, the person either recovers, or the disease (if left untreated) becomes more severe - toxic. Its occurrence is due to the systemic effect of diphtheria toxin on the body, which is secreted by the diphtheria bacillus.

This form of the disease is severe with pronounced symptoms:

• High body temperature – 39.5–41 0C.
• Strong headache.
• Paleness of the skin.
• Swelling of the tonsils, difficulty breathing.
• Soreness and enlargement of lymph nodes.

Serum dosage for toxic diphtheria is presented in the table below:

The injection is carried out through intramuscular and intravenous administration.

The specified amount of serum is administered once. If necessary, the same dose of the drug is administered after 24 hours. In severe forms of the disease, the total dose can be increased to 90,000-120,000 IU, and sometimes to 150,000 IU.

With the development of toxic shock, large hemorrhages caused by blood incoagulability (DIC syndrome) and other disorders, 2/3 of the dose is immediately administered to the sick person, and ¾ of the total dosage is administered over the course of 24 hours. Along with the serum, the patient is prescribed antibiotics for 5-7 days.

They do not inhibit diphtheria toxin, but help destroy dangerous microorganisms that complicate the course of diphtheria.

Equine anti-tetanus serum, purified, concentrated

Dosage regimen and method of administration

Emergency prevention of tetanus: Emergency prevention of tetanus involves primary surgical treatment of the wound and the creation, if necessary, of specific immunity against tetanus. Emergency specific prophylaxis of tetanus is carried out when:

• injuries with violation of the integrity of the skin and mucous membranes;
• frostbite and burns of the second, third and fourth degrees;
• out-of-hospital abortions;
• childbirth outside medical institutions;
• gangrene or tissue necrosis of any type, abscesses;
• penetrating injuries of the gastrointestinal tract;
• animal bites.

For emergency specific prevention of tetanus, the following is used:

• AS-anatoxin;
• human tetanus immunoglobulin (HTI);
• in the absence of ICHPS - antitetanus serum.

AS-toxoid and ICHPS are administered in accordance with the instructions for use of these drugs.
The scheme for selecting prophylactic agents for emergency specific prophylaxis of tetanus is presented in Table 1.

For the purpose of emergency prevention of tetanus, antitetanus serum is administered subcutaneously at a dose of 3000 IU.

Before administering antitetanus serum, an intradermal test is performed with purified horse serum diluted 1:100. To perform the test, use syringes with a graduation value of 0.1 ml and thin needles. The diluted serum is injected intradermally into the flexor surface of the forearm in a volume of 0.1 ml. The reaction is recorded after 20 minutes.

The test is considered negative if the diameter of the swelling or redness that appears at the injection site is less than 1 cm. The test is considered positive if the swelling or redness reaches a diameter of 1 cm or more.

If the intradermal test is negative, antitetanus serum is administered subcutaneously in an amount of 0.1 ml (use a sterile syringe, cover the opened ampoule with a sterile napkin). If there is no reaction, after 30 minutes, using a sterile syringe, the entire prescribed dose of serum is administered subcutaneously (for prophylactic purposes), intravenously or into the spinal canal (for therapeutic purposes).

If the intradermal test is positive or an anaphylactic reaction occurs to a subcutaneous injection of 0.1 ml of antitetanus serum, its further administration is contraindicated. In this case, the introduction of ICHPS is indicated.

The administration of the drug is registered in the established registration form, indicating the date of vaccination, dose, manufacturer of the drug, batch number, reaction to the administration of the drug.

Treatment of tetanus: Antitetanus serum is administered to patients as early as possible from the onset of the disease at a dose of 100,000-200,000 IU.

The serum is injected intravenously or into the spinal canal after testing sensitivity to a foreign protein (test with purified horse serum diluted 1:100). Depending on the severity of the disease, the administration of serum is repeated until the reflex seizures disappear.

Table 1 SCHEME FOR SELECTION OF PREVENTIVE DRUGS WHEN CARRYING OUT EMERGENCY SPECIFIC PREVENTION OF TETANUS

• PSS – antitetanus serum
• ICHPS – human antitetanus immunoglobulin

Notes:

1. Instead of 0.5 ml of AC toxoid, ADS-M toxoid can be used if vaccination against diphtheria with this drug is necessary. If the localization of the wound allows, it is preferable to administer AC toxoid to the area where it is located by subcutaneous injection.
2. Use one of the following drugs: IPSC or PSS (it is preferable to administer IPSC).
3. For “infected” wounds, 0.5 ml of AS toxoid is administered if 5 or more years have passed since the last revaccination.
4. The full course of immunization with AC toxoid for adults consists of two vaccinations of 0.5 ml each with an interval of 30-40 days and revaccination after 6-12 months with the same dose. According to the abbreviated scheme, the full course of immunization includes a single vaccination with AC toxoid in a double dose (1 ml) and revaccination after 6 months - 2 years with a dose of 0.5 ml of AC toxoid.
5. Two vaccinations according to the regular immunization schedule (for adults and children) or one vaccination according to the abbreviated immunization schedule for adults.
6. For “infected” wounds, IPS or PSS is administered.
7. All persons who received active-passive prophylaxis must be revaccinated with 0.5 ml of AC toxoid to complete the course of immunization after 6 months - 2 years.
8. If necessary, prescribe AC ​​toxoid to children under 6 years of age, the drug should be administered intramuscularly.
9. After normalization of the post-traumatic state, children under 3 years of age - in accordance with the terms of the National Calendar of Preventive Vaccinations.

How long does the vaccine last?

The introduction of serum according to Bezredko allows you to minimize the development of complications after the entry of a foreign protein into the body. The therapeutic dose is usually administered once - with proper and timely treatment, it allows you to cope with the disease.

But having experienced diphtheria and tetanus does not guarantee that a person will not get sick with them again. Vaccinations do not provide such protection against diseases. But the developed anti-diphtheria and anti-tetanus antibodies through vaccination protect the human body from the penetration of pathogens.

Therefore, these diseases either do not have time to develop, or will occur in a mild form, without causing complications.

The duration of action of vaccinations is directly related to the age at which a person was vaccinated:

• Children of the 1st year of life receive 3 vaccinations against diphtheria and tetanus (at 3 months, 4.5 months and six months) with a break between them of at least 45 days.
• At 1.6 years old, the 1st revaccination is given.
• 6-7 years – 2nd revaccination.
• 14 years – 3rd revaccination.
• For children over 18 years of age and adults, revaccination is done every 10 years from the date of the previous vaccination.

If a person was not vaccinated as a child, then in order to form specific immunity he needs to receive 3 vaccinations. If the planned vaccination schedule is violated or if more than 20 years have elapsed since vaccination, a person must be vaccinated twice with an interval of 40 days between vaccinations.

Correct administration of medicinal drugs according to the Bezredo method minimizes the risk of allergic reactions and prevents the occurrence of anaphylactic shock if all rules and algorithm of actions are followed.

TESTS

Description

Transparent or slightly opalescent liquid with a yellowish tint, without sediment. The determination is carried out visually.

Authenticity

The serum must neutralize the effect of tetanus toxin. The determination is carried out according to the section “Specific activity”.

Transparency

The optical density indicator should not exceed 0.05. The determination is carried out by the photometric method at a wavelength of 540 nm in a cuvette with a layer thickness of 3 mm, unless otherwise specified in the regulatory documentation.

Chroma

The optical density indicator should not exceed 0.15. The determination is carried out by the photometric method at a wavelength of 400 nm in a cuvette with a layer thickness of 3 mm, unless otherwise specified in the regulatory documentation.

Mechanical inclusions

Visible mechanical inclusions must comply with the requirements of the General Pharmacopoeia Monograph “Visible mechanical inclusions in dosage forms for parenteral use and ophthalmic dosage forms.”

pH

From 6.8 to 7.2, unless otherwise indicated in the regulatory documentation. The determination is carried out by the potentiometric method in accordance with the General Pharmacopoeia Monograph “Ionometry”.

Protein content

From 8 to 12%, unless otherwise specified in the regulatory documentation. The determination is carried out using the colorimetric method with a biuret reagent in accordance with the General Pharmacopoeia Monograph “Determination of Protein”.

Sterility

Must be sterile. Tests are carried out using direct seeding or membrane filtration methods in accordance with the General Pharmacopoeia Monograph “Sterility”.

Pyrogenicity

Must be pyrogen-free. The determination is carried out in accordance with the General Pharmacopoeia Monograph “Pyrogenicity”, unless otherwise specified in the regulatory documentation. Indicate the permissible limits for changes in animal body temperature and the test dose. Unless otherwise indicated, administer 1 ml of undiluted serum per 1 kg of rabbit weight.

Abnormal toxicity

Must be non-toxic. The determination is carried out in accordance with the General Pharmacopoeia Monograph “Anomalous toxicity”, unless there are other instructions in the regulatory documentation.

Specific activity

At least 1200 m international units (IU) in 1 ml. The specific activity of anti-tetanus serum is determined in the tetanus toxoid neutralization test using the Ehrlich method and is expressed in IU/ml (1 IU of tetanus antitoxin is the specific neutralizing activity of the antitoxic serum against tetanus toxin, which is contained in a certain amount of the international standard sample, which is anti-tetanus lyophilized horse serum).

Determination of a test dose of tetanus toxin. An experimental dose of tetanus toxin (L+/100) is the smallest amount of toxin that, when mixed with 0.01 IU of antitetanus serum when administered to mice in a volume of 0.4 ml, causes the death of 50% of mice (with symptoms of tetanus) on 4 days.

Several dilutions of tetanus toxin are prepared, differing by 10–20%.

When determining the experimental lethal dose of tetanus toxin, a standard sample (RM) of antitetanus serum activity, calibrated in IU in relation to the corresponding international standard sample, is used. CO is diluted with 0.9% sodium chloride solution to a concentration of 0.1 IU/ml. Mixtures are prepared consisting of 1 ml of CO activity of antitetanus horse serum, 2 ml of 0.9% sodium chloride solution and 1 ml of one of the dilutions of tetanus toxin. Mixtures of toxin with serum are carefully mixed and incubated at a temperature of (37 ± 1) °C for (45 ± 1) minutes, then 0.4 ml is injected into 4 white mice weighing (17 ± 1) g under the skin of the thigh. The animals are observed for 4 days. A dilution of the toxin is noted, which, when mixed with CO activity of the antitetanus serum, caused the death of 50% of animals from tetanus.

Determination of specific activity (titer) of antitetanus serum. Based on the expected activity, the serum is diluted with 0.9% sodium chloride solution to a concentration of 0.1 IU/ml. Several dilutions are prepared, differing from each other in activity by 10–20%.

1 ml of each dilution of the test serum is transferred into vials, 2 ml of 0.9% sodium chloride solution and 10 experimental doses of tetanus toxin in a volume of 1 ml are added. The resulting mixtures are carefully mixed, avoiding foaming, and after keeping at a temperature of (37 ± 1) ºС for (45 ± 1) minutes, they are administered to 4 white mice weighing (17 ± 1) g under the skin of the thigh in a volume of 0.4 ml.

The experiment is accompanied by control of the experimental dose of the toxin, for which a mixture is prepared containing 1 ml of CO activity of antitetanus serum, diluted to a concentration of 0.1 IU/ml, 1 ml of toxin containing 10 experimental doses, and 2 ml of 0.9% sodium chloride solution. The mixture is incubated under the same conditions as the test serum, and it is administered to 4 white mice weighing (17 ± 1) g under the skin of the thigh in a volume of 0.4 ml. The animals of the experimental and control groups are observed for 4 days, noting the number of mice that died from tetanus.

The specific activity (titer) of the serum is calculated based on its highest dilution, which, when mixed with a test dose of the toxin, provides 100% protection from tetanus in mice. The test is not taken into account if all mice in the control group remained alive without signs of tetanus.

Specific activity

At least 1000 IU per 0.1 g of protein. Specific activity (X) is calculated using the formula:

Where:

T – serum titer, IU/ml;

C – protein concentration, g/ml;

10 – constant coefficient.

Sulfate ions

No more than 0.025%. Determination is carried out using the colorimetric method. To 5 ml of the test sample and 5 ml of the working standard solution, add 0.5 ml of a 5% barium chloride solution and mix. After 15 minutes, the samples are mixed and the optical density of the suspensions is measured at a wavelength of 540 nm in cuvettes with a layer thickness of 10 mm against a control solution containing 5 ml of sample and 0.5 ml of purified water; for the reference solution - purified water (5 ml).

The test is carried out in 2 repetitions. The average value is used for calculation.

The percentage of sulfate ions (X) is calculated using the formula:

Where

Aexperience – the value of the optical density of the test sample;

Aethalon is the optical density value of the working standard solution.

Notes.

1. Preparation of a stock solution of potassium sulfate (1 mg/ml sulfate ions). In a volumetric flask with a capacity of 1000 ml in purified water, dissolve 1.8140 g of potassium sulfate, dried to constant weight at a temperature of 100 - 105 ° C, adjust the volume of the solution with water to the mark and mix. The solution is stored at room temperature for 1 year.
2. Preparation of a working reference solution of potassium sulfate (0.002% sulfate ions). Place 1 ml of a basic solution of potassium sulfate into a 50 ml volumetric flask, add purified water to the mark and mix. The solution is used freshly prepared.

Sodium chloride

From 0.85 to 0.95%. The determination is carried out in accordance with the General Pharmacopoeia Monograph “Quantitative determination of chlorides by reverse precipitation titration in immunobiological medicinal products.”

Chloroform

No more than 0.1%. The determination is carried out using a colorimetric method based on the ability of chloroform to form a compound with a quinoid structure with resorcinol in an alkaline environment, which gives a color reaction.

Add 0.1 ml of the test sample and the reference sample (0.1% chloroform solution) into the test tubes, add 0.9 ml of 0.9% sodium chloride solution, 2 ml of 20% sodium hydroxide solution, 1 ml of 10% resorcinol solution and stir. The contents of the test tubes are mixed and heated in a boiling water bath for 1 minute. The samples are carefully cooled to a temperature of 15 - 18 0C, then the optical density of the colored solution is measured at a wavelength of 540 nm in a cuvette with a layer thickness of 5 mm in comparison with a control solution consisting of 1 ml of purified water, 1 ml of 0.9% sodium chloride solution and 2 ml of 20% sodium hydroxide solution. The resorcinol solution is not added to the control solution, because its oxidation products turn green.

Calculation of the chloroform content is carried out by comparing the optical density of the test sample and the reference sample (0.1% chloroform solution).

The percentage of chloroform (X) in the test sample is calculated using the formula:

Where:

Aisp is the optical density value of the test sample;

Ast – the optical density value of the reference sample – 0.1% chloroform solution.

Notes.

1. Preparation of 0.1% chloroform solution. Place 0.1 ml of chloroform in a 100 ml volumetric flask, adjust the volume of the solution to the mark with purified water and mix. The solution is used after 24 hours.
2. Preparation of a 10% resorcinol solution. Place 5 g of resorcinol in a 50 ml volumetric flask, dissolve it in 30 ml of purified water, adjust the volume of the solution to the mark with the same solvent and mix. The solution is used freshly prepared.

Recoverable volume

No less than nominal. The determination is carried out in accordance with the General Pharmacopoeia Monograph “Extractable volume of dosage forms for parenteral use.”

Packaging and labeling

In accordance with the General Pharmacopoeia Monograph “Immunobiological medicinal products”.

Transportation and storage

At temperatures from 2 to 8 ºС, unless otherwise specified in the regulatory documentation. Freezing is not allowed.

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