CS201347B1 - Production of monospecific pr ecipitation serums for diagnostical purposes - Google Patents

Description

The invention relates to the production of monospecific precipitated sera for diagnostic purposes, in which a method for isolating immunoglobulin synthesized by a specific immunoadsorption method for a particular bacterial particle antigen and a method for immunizing experimental animals used for production is provided.

Analysis of the prior art shows that monospecific precipitated sera contain antibodies (precipitates) that specifically bind to and after binding of the protein species. ba precipitates in the insoluble complex. Precipitation sera are produced against different antigens, mainly against individual blood serum proteins. Since these sera only precipitate the antigen that prompted their production, they are suitable for qualitative and quantitative detection of the respective antigen in different body fluids using immunochemical reactions. Such reactions are, for example, bi-directional immunodiffusion methods according to Ouchterlony, immunoelectrophoresis, simple, radial immunodiffusion and the like. In addition, these precipitating sera are an essential raw material in the production of fluorescent sera for direct and indirect fluorescence.

Methods using monospecific precipitated sera are among the newer advanced immunodiagnostic methods, nowadays indispensable, either in research or in conventional veterinary immunodiagnostics. Of great theoretical and practical importance is the qualitative and quantitative evidence of blood serum immunoglobulins. For the production of monospecific precipitation sera is

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201 347, it is necessary to obtain, first of all, individual classes of immunoglobulins (e.g. IgG and IgM) in an immunochemically pure state. For the isolation and purification of various classes of immunoglobulins, relatively complex physicochemical methods have been developed over the last decade using chemicals and instruments largely from imports from the capitalist states. Since the literature on this subject is very extensive and basically is based on the same principles, I mention some of them used to isolate IgG and IgM class immunoglobulins.

S e e £ e (Allegemeine und Experelle Immunologie und Immunopathologie sowie ihre klinische Anwendung, - Georg Thieme Verlag - Stuttgart 1968, page 564), uses the following methodology to isolate IgM immunoglobulin:

To 10 ml of blood serum is added 0.4 ml of a 10% solution of Dextran sulfate dissolved in distilled water and 1 ml of 1 M CaCl2 solution. After mixing, the solution was allowed to stand at room temperature for 15 minutes. The precipitated lipoproteins were removed by centrifugation (10 min at 6,000 g). The supernatant was dialyzed briefly against distilled water and then made up to 200 ml with 0.75% boric acid solution. After 30 min. After standing at room temperature, the precipitate formed is centrifuged (10 min. at 2,000 g). The sediment is washed twice more with the boric acid solution at the indicated concentration. After washing, the precipitate is dissolved in 3-4 ml of the following buffer: Tris-Hydroxymethylaminomethane 7.96 g, NaCl 58.45 g is dissolved in distilled water containing 0.033 M HCl (pH 8.0).

The precipitate thus dissolved is then applied to a column packed with 2.8 x 80 cm Sephadex G-200 gel fixed on said Tris buffer. The first peak fractions contain pure IgM immunoglobulin. However, the method does not exclude alpha, macroglobulin contamination, which is found in almost all IgM immunoglobulin preparations prepared by the method. Alpha-macroglobulin has very close physico-chemical properties to IgM immunoglobulin.

F e y (Methods of Purification and Quantitation of Bovine Immunoglobulins, Zbl.Vet.Med.

8,23 (1976, 269-300) discloses the following method for isolating IgM immunoglobulin:

Blood serum is dialyzed against 0.01 M phosphate buffer pH 5.4. The Euglobulin precipitate formed during dialysis is washed twice in 0.01 M acetate buffer containing 0.15 M NaCl, pH 5.4, and dissolved. The amount of acetate buffer should be 20 µl of the original amount of serum. Thereafter, with stirring, 0.1 M ZnSO4 was added dropwise to a final concentration of 25 mM. The resulting precipitate was stirred at room temperature for two hours and then removed by centrifugation at 3000 rpm. A supernatant is used from which the Zn ions are removed with 1 N tetrasodium EDTA solution. After concentrating the solution, this is placed on a Sephadex G-200 gel column fixed on 0.1 M Tris-HCl buffer containing 0.1 M NaCl, pH 8.6. The first peak fractions contain IgM immunoglobulin. However, only the fractions found in the ascendent part of the first peak are used for immunization of animals. However, even the precipitated sera thus prepared contain precipitates against IgG and alphagM protein, which need to be removed from the precipitated serum. N. alpha-M globulin removal prepared an isolation method to obtain said globulin in a pure state. This further complicates the described method.

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Almost all authors use gel filtration (e.g. Sephadex G-200) and ion exchangers (such as DEAE cellulose and DEAE Sephadex) to isolate the IgG class of immunoglobulin. For this purpose, either whole blood serum or colostrum, or isolated immunoglobulins therefrom in the non-green state, using ammonium sulfate, sodium sulfate or rivanol, is used.

L e v i e x (immunoglobulins Bovines et Brucellose, Ann.Rech.Veter., 197 ^, 5,329-342) gives the following procedure for the isolation of IgG2 immunoglobulin:

Whole bovine serum is filtered on Sephadex G-200 on a 90x5 cm column fixed on 0.1 M Tris-HCl buffer containing 0.15 M NaCl and sodium azide at a total concentration of 1: 5000 at pH 8.0. Second peak fractions containing IgG immunoglobulin and other proteins of similar molecular weight are further fractionated on DAEA cellulose using the following buffers: 0: 0.01 M pH 8.0 - 1: 0.015 M pH 7.3 - 2: 0.03 M pH 7.3-3: 0.06 M pH 7.0-4.0 M pH 6.0. Uses phosphate buffer. The solution obtained by gel filtration dialyzed against 0.01 M phosphate buffer and at this molarity the fractions recovered were put on said ion exchange and filtered at the same molarity. In this way, pure IgG2 obtains an immunoglobulin. Other authors use more or less complicated methods essentially identical and differ only in that they do not proceed from fractionation from whole serum, but first isolate IgG immunoglobulin in a non-fatal state using exchanged chemicals, whether from blood serum or colostrum and thus obtained immunoglobulin fractionates on gels or ion exchangers.

The second prerequisite for the production of high-quality monospecific precipitation sera is the choice of a suitable animal. Kings, goat, donkey and pigs are most commonly used for this purpose. An important limiting factor is also the immunization procedure (site of application, interval between antigen applications, antigen doses, use or non-use of various adjuvants, etc.). Immunization procedures are reported very briefly in the literature as they are commercially manufactured preparations whose precise manufacturing process is secret. As a rule, immunization procedures are time-consuming and do not always guarantee the precipitated sera of the desired quality. By way of illustration, I provide an immunization procedure for obtaining an antiserum against animal gamaglobulin. The authors are Semotan et al. Final Report „Conjugates, Bioveta, Ivanovice na. Hané 1974.

On the first day, 3 ml of Freund's adjuvant is administered subcutaneously. Next, an intravenous solution of 1 ml of gamaglobulin is administered for 9, 12, 15, 19, 21, 26, 307 and 33 days. On J6, the day is again administered 3 ml of Freund's adjuvant subcutaneously. On days 44, 47 and 50, 1 ml of a 2% gamaglobulin solution is administered again intravenously. On day 53, the adjuvant is again administered in the indicated amount subcutaneously. The immunization procedure is terminated by intra-venous administration of 3% gamma globulin at 60, 63. and 66.day.

As a result, relatively complicated methods using expensive chemicals and instruments are used to isolate individual classes of immunoglobulins, while not guaranteeing protein isolation of sufficient purity to immunize animals. In addition, sophisticated immunization procedures are used which do not always guarantee the production of precipitated sera of the desired quality. Accordingly, the production of monospecific precipitation sera requires further improvement.

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The essence of the invention consists in the practical use of own work, research results obtained in the detection of the dynamics of antibody production in brucellosis of livestock, in immunization or in the course of infectious process. In the immunization of animals at an early stage of the immunization process, almost all antibodies are bound exclusively to the IgM class, at a later stage the antibody activity is bound to the IgG class. It has also been found in our own experiments that brucella antibodies bound to the IgM class are relatively more thermoresistant than brucella antibodies bound to the IgG class. IgM class antibodies are completely destroyed for 10 min. by heating to 70 ° C, wherein IgG class antibodies are almost intact. These two moments were used in the production of monospecific animal anti-IgM and anti-IgG precipitation sera. Thus, isolation of IgM immunoglobulins can be accomplished by immunoadsorbent method using a brucella antigen using brucella positive serum from immunized animals for 5 to 6 days from the start of immunization. However, to isolate IgG class immunoglobulins, blood serum from immunizing animals 14 to 15 days after immunization should be used, wherein IgM-class bound antibodies are removed by inactivation in a water bath at 70 ° C for 10 minutes. Antibodies bound to the surface of the brucella antigen, now as an antigen, serve to produce the appropriate antiserum upon administration to suitable test animals. An immunization procedure was chosen such that a small amount of immunoglobulins bound to the surface of the brucella antigen was sufficient for the abundant production of antibodies. It should be noted that the brucella antibodies of the appropriate class are so tightly chemically bound to the brucella corpuscular antigen receptors that the other serum proteins are completely removed by centrifugation (washing). IgG and IgM classes are immuno-chemically bound to the brucella antigen surface using appropriate brucella sera. The immunoglobulins thus obtained together with the corpuscular brucella antigen are used to immunize rabbits. An immunization procedure is chosen which, even with low amounts of IgG and IgM class immunoglobulins, guarantees the production and thus production of high-quality monospecific precipitation sera in a relatively short time.

A method for producing monospecific precipitation sera for diagnostic purposes is characterized in that a corpuscular bacterial antigen, preferably brucellus, is used to immunize animals, preferably rabbits, for slow agglutination Specifically immunoadsorpted by antibodies of different classes of immunoglobulins, which is administered perfectly washed (at least 3 x) preferably in a 0.85 saline solution of sodium chloride. In an immunization process, preferably with complete Freund's adjuvant, subcutaneously (between the toes and no limbs) and intramuscularly, falls into a vein without an adjuvant.

DESCRIPTION OF THE INVENTION

1. Preparation of brucella positive serum containing IgG and IgM antibodies:

Large animals (bovine, horse) were injected intra-venous on the first and second day with 1 ml of brucella antigen for slow agglutination. The immunization procedure is similar in smaller animals (pig, sheep, goat) with the difference that only 2 ml of antigen is applied. to day 15 for the production of anti-IgM precipitation sera. Collect the collected blood and centrifuge at 2000 g for 30 minutes to obtain clear serum. If the serum is not processed immediately, it is preserved by lyophilization or freezing at -20 ° C. The serum so preserved is usable for the above purposes for 2 years.

2. Preparation of brucella antigen sensitized with IgM and IgG class antibodies:

Production of anti-IgM precipitation serum:

For the production of monospecific anti-IgM precipitating serum, serum from the respective species, which we collected 5 to 6 days after the first antigen application, is used positively. The serum is inactivated at 60 ° C for 30 min. in a water bath. Serum is assayed for brucella antibody levels by tube agglutination according to valid examination methods.

In order to achieve the best possible occupancy of all brucella antigen receptors, an 8-16-fold amount of brucella antibodies that are capable of agglutinating all the brucellus (agglutination to ++++) in slow agglutination is used. For example, if the serum has a titer of 10O, this means that one unit of brucella antigen (0.05 ml) is agglutinated with serum at a 1: 16O dilution. In this case, half or the same amount of positive brucella serum is used per antigen unit. Serum is in required! ratio is mixed with brucella antigen and incubated in a thermostate at 37 ° C for 4 to 8 h and then overnight in a refrigerator. ,. At + 4 to + 6 C, the antigen is fully populated with antibodies if the antigen supernatant still contains brucella antibodies after centrifugation, i.e., it agglutinates the brucella antigen in rapid agglutination on a slide. If this is not the case, serum is added to the brucella antigen and incubation is performed in the same manner as before.

The Brucella antigen is then centrifuged for 10 min, at 2000-g and the sediment is washed 4 times in saline NaCl in the original volume. After the last centrifugation, the sediment is resuspended in the amount of phenol-physiological solution (phenol content 0.5%), which amount of the antigen we used for flood sensitization. Shake the antigen with sterile beads. Thus sensitized antigen stored at +4 to +8 ° C is useful for immunizing rabbits for at least one year.

Production of anti-IgG precipitating serum:

No, blood serum collected at 14-15 days after immunization of the respective animal species is used to produce monospecific anti-IgG precipitating serum. Serum is diluted in equal parts with 0.85% NaCl solution and inactivated in ultrathermostate for 10 min. at 70 ° C. With such inactivation, serum generally loses its agglutination ability or induces zoning, i.e. that agglutination does not occur where the largest amount of antibodies is found, but in higher facilities. With this inactivation, IgM antibodies were completely removed and only antibodies bound to the IgG class of immunoglobulins remained. However, the titer of antibodies can only be substantiated by the Coombs antiglobulin technique in its own modification.

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For antigen sensitization, we use 8 to 16 times the amount of antibodies per unit of antigen (0.05 ml) that is able to induce agglutination at ++++ in a modified antiglobulin assay. For example, if the serum has a titer of 640, 2 or 4 ml of brucella serum inactivated in the same manner are used per ml of brucella antigen. When determining the brucella brucella antibody titer in the modified antiglobulin test, it should be remembered that dilute serum is used. Washing of the antigen is done in a similar way to the preparation of sensitized IgM antigen by antibodies, except that it is centrifuged for 3θ minutes at 6000 g. The applicability of this antigen is consistent with the foregoing.

3. Immunization of rabbits for the production of monospecific anti-IgG and an ± 1 * IgM precipitation sera for single species:

The immunization procedure is simple for both classes regardless of the immunoglobulin class. Rabbits weighing 4 to 6 kg are used for immunization. The first immunization of rabbits is carried out to the skin of the fingers between each of the front and hind legs so that the two locations of each leg applied after 0.25 ^m l of the antigen. The antigen for the first and second immunizations is prepared with complete Freund's adjuvant by adding 1 ml of Freund's adjuvant to 1 ml of brucella sensitized antigen (the antigen must be mixed thoroughly as it settles on standing) and homogenizing the brucella suspension with the adjuvant by syringe. About ten times the contents are stretched and vigorously ejected from the syringe. For the first immunization, there is a volume of two ml per rabbit (1 ml brucella antigen and 1 ml adjuvant). The second immunization of rabbits is performed intramuscularly in the femoral muscle on day 14 with the same antigen as the first immunization at a dose of 1 ml. For the third time, an antigen without adjuvant is applied on day 21 into the ear marginal vein at a dose of 1 ml per rabbit and on day 22. the same antigen (without adjuvant) is also administered at a dose of 1 ml per rabbit.

Rabbits are bled for 5 days after the last antigen administration and serum is obtained by allowing the blood to coagulate. After separation of the blood cake from the wall of the centrifuge cup, it is placed in a thermostat for 1 hour. Then the blood is centrifuged for 30 min at 3000 g and the clear serum is withdrawn by syringe.

Precipitating sera are examined immunoelectrophoretically against whole blood serum or starting body fluid of the respective healthy animal species. We will receive only one distinct precipitaine line characteristic of the immunoglobulin class. However, the differences are more marked when the precipitation sera are examined for precipitation activity. This is demonstrated by a simple radial immunodiffusion method by incorporating the appropriate antigen (immunoglobulin) into the gel (agar or agarose) and adding the same amount of precipitating sera to the holes. The precipitating serum is of higher quality, resulting in a larger and more sharply defined precipitation circle. in the same time. Controlled by this method for the precipitation sera produced according to the invention, exhibit up to two to five times greater precipitation activity than other methods for the production of precipitation sera. Moreover, it is not neglected

201 347 the advantage of the proposed production and that the precipitating sera are produced in a shorter time and there is no need to use complicated isolation methods to isolate and purify immunoglobulins of different classes to get them in an immunochemically pure state. The higher efficacy (precipitation activity) is explained by the fact that the test animals have poorly formed antibodies (precipitins) against pure immunoglubulins, but relatively good are antibodies against impurities (another type of protein) which they may contain, even in small amounts. However, the carrier of immunoglobulins is much more potent antigen for the test animals, which then advantageously forms precipitates against them. However, the potentiating effect of the bacterial carrier itself, in particular of the polysaccharide content, on the immune system of the test animal cannot be neglected. This results in the formation of more precipitates in a shorter time in immunized animals, which is the essence of the invention.

Claims (1)

OBJECT OF THE INVENTION A method for producing monospecific precipitation sera for diagnostic purposes, characterized in that animals, preferably rabbits, are immunized with a corpuscular bacterial antigen, preferably brucella, for slow agglutination, which is specifically immunoadsorbed by antibodies of different classes of immunoglobulins, said antigen at least three times washed with physiological saline and for the first and second immunization of the animals, the antigen is mixed with complete Preund's adjuvant and applied subcutaneously between the toes and no limbs and intramuscularly and for the third immunization the non-adjuvanted antigen is injected intravenously and subsequently animals are bled and serum coagulation by removing the blood cake.