RU2010577C1 - Method for preparation of diagnostic serum - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: method involves immunization of animals with bacterial antigens introduced into lymphonodi and intramuscularly with solution of feracryl followed by drawing blood, separating immune serum forming an antigen-antibody complex and intravenous immunization with said complex. Time for preparation of horses producing tularemia diagnostic serum is reduced from 2.5-3 years to 2-3 months. Rabbits producing brucellosis agglutinating serum are prepared with inactivated antigens instead of live virulent cultures of brucellosis causative agents which rules out the necessity for strict observance of antiepidemic conditions. Sera prepared according to claimed method can be utilized for producing labelled gamma-globulins. EFFECT: higher efficiency.

Description

 The invention relates to biotechnology, in particular to the production of diagnostic preparations, in particular for the production of highly active immune sera during immunization of animal producers with various antigens.

 A known method for the production of diagnostic tularemia serum, which consists in repeated intravenous hyperimmunization of horses producing corpuscular bacterial antigen [1].

A known method of obtaining diagnostic brucellosis agglutinating serum, which consists in immunizing rabbits producing live virulent Br cells. abortus 146 and Br. melitensis 565 (1.10 ⁹ m.k. in 1 ml) four times. The first three injections of antigen in a volume of 0.5; 0.75 and 1.0 ml (in a mixture with an equal volume of Freund's complete adjuvant) are injected subcutaneously at several points along the spine, a fourth (1 ml) - intravenously. The intervals between the first and second, second and third injections are one, between the third and fourth - two to three weeks. Animals are totally bled two weeks after the final injection of antigen. Diagnostic brucellosis agglutinating serum has a titer of at least 1: 1600 [2].

 There is also a method of obtaining immune sera for a test system diagnostic for pathogens of zoonoses: plague, glanders, melioidosis, tularemia, anthrax using the indirect immunofluorescence method. Rabbits weighing 2.5-3 kg are immunized intradermally multipoint along the back with a mixture of plague pathogen antigens (0.2 mg), glanders (1 mg), melioidosis (1 mg), brucellosis (3 mg), tularemia (3 mg) with Freund's adjuvant hyperimmunization consists of 4-5 injections at intervals of 6 days. After 30 days, a second hyperimmunization cycle is carried out according to the above scheme. On the 7th day the animals are bled. The activity of sera in the indirect immunofluorescence reaction (RNIF) 1: 256 [3].

 The disadvantages of the known methods are that the methods do not provide highly active serum, time-consuming and time-consuming. The training of producing horses requires the expenditure of more nutrient media, working and biological time. When using a live virulent culture of the causative agent of brucellosis as an antigen, it is necessary to strictly observe the anti-epidemic mode of operation.

 The closest in technical essence is a method for producing tularemia diagnostic serum. Production horses are hyper-immunized with a tularemia microbe culture killed by boiling. Hyperimmunization is carried out in cycles consisting of 5-6 intravenous injections in increasing doses at five-day intervals. Such injections are carried out up to 17 or more.

 The disadvantages of this method: it takes 2.5-3 years of painstaking work before you can get the right titer of serum (1: 3200).

 The aim of the invention is to reduce the preparation time of animal producers and increase the specific activity of the target product.

 The goal is achieved by the fact that animals are immunized with antigens in the lymph nodes and intramuscularly with Feracryl solution, blood is taken, serum is isolated, antigen is added, the antigen-antibody complex is obtained and the cycle is administered intravenously.

 PRI me R 1. Obtaining tularemia diagnostic serum. Production horses are injected subcutaneously in the region of the lower third of the neck with 2 ml of a 3% aqueous-alcoholic solution of feracryl. On the 5-7th day, 300 billion m of cu. Are injected with formalin-inactivated tularemia microbe culture into the pre-scapular lymph nodes mixed with 2 ml of a 1% solution of feracryl. After 3 days, the same mixture is administered intramuscularly in increasing doses (400, 500, 600, 700 billion m. K.) with an interval of 4 days. On the 7-8th day after the last injection of the antigen, a blood sample is taken, serum is obtained and the antibody titer is determined. If the antibody titer is lower than 1: 3200, they do stimulating bloodletting 2-3 l. Get immune serum. Four servings of serum are taken in 100 ml each, of which an antigen - inactivated tularemia microbe culture is added to each of the above increasing doses and a second immunization cycle of four intravenous injections with 4-day intervals of the antigen-antibody complex (AG-AT) is prescribed.

 On the 7-8th day, a blood sample is taken, immune serum is obtained, and the antibody titer is determined. It is sufficient (1: 3200) for industrial bloodletting. Thus, the training period for producing horses is reduced from 3 years to 2-3 months.

 PRI me R 2. Obtaining diagnostic brucellulose agglutinating serum.

To production rabbits weighing 2.5-3 kg, 0.6 ml of a 3% aqueous-alcoholic solution of feracryl is introduced into the pads of the hind legs. On the 5-7th day, the lipopolysaccharide antigen (LPS) Br. melitensis 16M in an amount of 1 mg is mixed with 1.2 ml of a 1% aqueous-alcoholic solution of feracryl and 0.6 ml are injected into the popliteal lymph nodes. After three days, the same mixture is administered intramuscularly three times at intervals of 4 days. On day 30, 4 ml of immune serum is obtained from animals, 4 mg of Br is added to it. melitensis 16M dissolved in 1 ml of LPS 0.9% sodium chloride. The AG-AT complex is prepared and administered four times 1 ml at intervals of 3-4 days intravenously to the same animal from which the immune serum was obtained. On day 7-8, animals are bled. The activity of sera in RID is 1: 32, in ELISA - 1: 7.10 ⁶ , in the volumetric agglutination reaction 1: 2560 - 1: 5120.

 PRI me R 3. Obtaining serum for the test system diagnostic to the causative agents of plague, glanders, melioidosis, brucellosis, tularemia.

 All operations associated with the immunization of producer rabbits are performed in the same sequence as in the preparation of brucellosis agglutinating serum. Pathogen antigens are injected in the complex in the following doses: plague 0.2 mg, glanders 1 mg, melioidosis 1 mg, brucellosis 3 mg, tularemia 3 mg. Half doses are used to form the AG-AT complex. The serum activity for the test system in RNIF 1: 512 - 1: 2048.

 The advantages of the proposed method: obtaining highly active serums; shortening the training of producers; strict adherence to the anti-epidemic regimen of work with animals is not required, since inactivated antigens are used during immunization. (56) Tularemia diagnostic dry serum for RA. Production regulations FS 42-112BC-88. Irkutsk Research Anti-Plague Institute.

 Dry serum diagnostic brucellosis agglutinating. Production regulations. Odessa enterprise of pharmaceuticals.

 Instructions for the manufacture and control of a diagnostic test system for the detection of pathogens of plague, glanders, melioidosis, tularemia, brucellosis, anthrax using the indirect immunofluorescence method. Research Anti-Plague Institute of the Caucasus and Transcaucasia.

Claims (1)

 METHOD FOR PRODUCING DIAGNOSTIC SERUM mainly against zoonoses, including immunization of animals with antigenic material, blood sampling and separation of serum, characterized in that the animals are immunized into the lymph nodes and intramuscularly, using the antigen mixed with Feracryl solution, and the antigen used for immunization is added to the separated serum , the resulting mixture is repeated intravenous immunization, blood is taken and the target product is separated.