JPH01500900A - Hemoglobin polymer complex and its preparation method - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Compositions and methods for immunological castration and ovarian removal This invention aims to suppress social and sexual behavior in male livestock and to suppress ovulation and ovulation in female livestock. and the suppression of estrous cycle, more specifically, the combination used for immunological castration and ovarian removal. Concerning compositions and methods.

Normal sexual behavior in female animals is maintained by androgens or male sex hormones; They are produced primarily in the testes. One of the main androgens is testosterone . For farmers, surgical castration eliminates sexual behavior in livestock and eliminates their aggressive habits. It is common to take a method of weakening the However, surgical castration has several It comes with some inconvenient problems. These include bleeding, infection, and stress-related weight loss. included (Schanbacher, 1984). In addition, castration reduces growth rate and Both the rate of fat and non-adipose tissue deposition are reduced (Lopertran et al., 1970).

An alternative to surgical castration is luteinizing hormone-releasing hormone (hereafter LHRH). active immunization against (indicated as ) or analogs thereof. LHRH is the hypothalamus is released into the pituitary portal vein. LHRH binds to gonadotropins in the anterior pituitary gland It is thought to stimulate the secretion and release of luteinizing hormone (LH).

LH acts on Leydig cells in the testis to increase testosterone production. L.I. (Active immunization against RH disrupts communication between the hypothalamus and pituitary gland.

The site where antibodies against LHRH can inhibit reproductive function is located in the portal vein of the pituitary gland. (Champachel, 1984).

Active immunization against LHRH significantly alters the function and morphology of the anterior pituitary gland. Change occurs. As a result, rats (Cock, 1977; Fraser et al., 198 2 years), rabbits (Arimura et al., 1974) and monkeys (Chappell et al., 198 Pituitary gland weight, gonadotropin content and L HRH binding is reduced. Additionally, testosterone secretion decreases and the testicles regress. (Rat, Fraser et al. 1982, marmoset monkey, Horazis and Bibburn, 1977, Ewe, Schanbacher et al., 1982, Shefcourt et al. , 1982, Bull, Robert Rann and others, 1979, 1981, 1 982, 1984).

Liveweight gain rates in bulls actively immunized against LHRH were lower than in early steers. It is also significantly faster (Lopartran et al., 1982). Immunized steers can also be castrated early. They convert food more effectively than cows and have a higher percentage of dressing out. The carcass has less fat, the height of the withers is short, and the girth is narrow ( Robert Ran et al., 1982).

Male calves that respond well to active immunization against LHRH are Fucault et al., 1982) and decreased secretion of testosterone, as well as testicular tissue. (Lono (-Tran et al., 1979, 1981, (1982, 1984). Subjective assessment of behavior indicates that immunized steers are male (non-castrated). They seem to be more similar to early steers, which are more docile and easier to handle than cows. and others, 1979, 1981, 1982, 1984). immunized When bulls are brought into contact with cows in heat or with artificial cows, they have high antibody titers. Three of the five animals did not attempt to mount, but the other two did and these animals In one case, ejaculation occurred (Robert Ran et al., 1982). In ongoing research , Robertran et al. (1984) found animals that responded well to immunization against LHRH. compared to non-immune subjects, riding, heading and licking found that immunocastrated animals were less active than (non-castrated) bulls in terms of movement and movement. Ta.

Active immunization against LHRH suppresses ovulation and estrus in the fetus. (Arimura et al., 1979), rabbits (Arimullah et al., 1979) 3 years), hamsters (De U Cruz et al., 1976) and rats (Fraser et al. - and Baker, 1978) was reported to cause gonadal atrophy. .

Clark et al. (1978) in sheep and Fraser et al. (1974) in rats. ) and McCormack et al. (1977) in rhesus macaques, anti-L HRH treatment resulted in a significant decrease in plasma LH concentrations. Sunawa Section Anti LH R) (Treatment is the same for females as for males for plasma LH concentration and gonadal function. seems to have the effect of High LI (febrile hands showing evidence of RH antibodies are indicative of anovulation) However, the study by Clark et al. (1 978) is particularly relevant.

Although active immunization against LHRH has been successful in male calves and other animals, , there was considerable variation between individual animals in their immune responses. most trials In experiments, male calves immunized against L) (RH) with high antibody titers Only about half responded satisfactorily to treatment. This is a 12 week old (Robert Rann) and others, 1979, 1981), 20 weeks of age (Robert Ran et al., 198 4 years) and immunized males at 28 weeks of age (Robert Ran et al., 1982). This was reported for calves. Chefcoat et al. (1982) is one blade. Golden male calves were actively immunized against LHRH, and antibody titers were determined twice in animals. It was reported that the level increased after the immunogen injection but started to decrease after the third injection. Ta. As a result, a fourth injection was required to recover the antibody titer.

The phenomenon of deficiency of immune response to antigenic stimulation is well documented in the literature. For example, Barba (1976) in a textbook on ``veterinergy immunology.'' ``In general, the antibody titers found in members of the vaccinated population are within statistical normality.'' curve, distributed into a small number of animals with high titers and another small group with no response ” on pages 199-200. In another section, “For quite a large number of Some vaccinated animals are unprotected when tested (by There was also. Errors in technique may explain some of these, but for others , no matter how many times the antigen is administered and in which other treatments are used to stimulate immunity. No matter what measures are taken (i.e., the use of adjuvants), the state of immunoprotection cannot be fully (achieved). I can't do that.''

LHRH is a small peptide consisting of 9 amino acids. In the field of immunology, small immunology It is well established that when injected into a host, it elicits an insufficient immune response. has been done. Therefore, in each of the above tests, L)(RH is defined as one specific carrier protein, e.g. chemically conjugated to human serum albumin.

Surprisingly, Applicants of the present invention have discovered that LHRH or analogs of LHRH have no less than two types. When an animal is immunized with a combination of conjugated carriers or more different carriers, We found that a synergistic interaction occurs. conjugated with carrier(s) Combinations of analogs of LHRH or LHR) (conjugate to a single carrier) in a higher proportion of animals than when administered LHRH or an analogue of LHRH. to elicit an effective immune response (measured in terms of antibody titer).

According to one aspect of the invention, an antibody specific for LHRH or an analogue thereof is administered to an animal. A composition intended for production in L. Compositions are provided that are individually conjugated to analogs of HRH or LHRH.

Analogues of LHRH (LHRH) (LHRH) are analogs of It is a peptide that has been Variations to the native sequence include the amino or carbon of LHRH. There is an addition of one or more amino acids to the carboxy terminus.

Types of livestock included within the scope of this invention include livestock, sheep, goats, cats, guinea pigs. There are primates, including pigs, dogs, reindeer, horses, and women.

Any type of carrier that is a useful immunogen may be used. Preferably a carrier more preferably the protein is soluble in aqueous solution, but is a precipitated protein. Quality can also be used.

Examples of preferred proteins include diphtheria toxoid (DT), human serum albumin (HS A), Staphylococcus protein A (P, A,,), Keyho Le linbet hemocyanin (KLH) and tetanus toxoid (TT) There is.

Generally, a combination of two or more different carriers may be used. Like Alternatively, the carrier is HSA and proteins such as P, A, or KLH and TT. Each of these proteins is then conjugated to LHRH. however, Particularly preferred is that each carrier conjugated to LHRH and administered to the animal separately is two or more carriers to elicit a good titer response. particularly desirable is a composition in which one of the carriers is diphtheria toxoid (DT).

Other carriers that can be used in this invention include ribopolysaccharides, polysaccharides, glycopeptides. , muramyl peptide analogs, peptidoglycan [from bacterial cell walls, liposomes] , lecithin-like substances, and purified protein derivatives from bacterial proteins, e.g. tuberculin. There is a conductor.

Conjugation of LHRH and LHRH analogs with various carriers involves conventional techniques. techniques may be used. For example, heterobifunctional agents such as 5PDP (Pharmacia, Viscataway, New Chassis), carbodiimide, glutaraldehyde The biotin/avidin or biotin/avidin systems are useful. LHRH or Conditions are generally selected that maintain the LHRH analog in its biologically active conformation. be done.

Preferably the composition includes an adjuvant. Examples of adjuvants that may be used include: Aluminum hydroxide, Freund's incomplete adjuvant, Freund's complete adjuvant DEAE dextran, levamisole, PCG and polyA1 polyC or has polyU. Particularly preferred adjuvants are those that do not cause local inflammation. It is quality. An example of such a substance is bacterial cell wall material such as peptidoglycan, or is a mineral oil composition containing a synthetic derivative of said cell wall material. Such synthetic substances are Known as mildipeptide.

The composition may optionally contain a buffer such as Tris-MCI2. hepes, pipes or other can be buffered to physiological pH using a suitable buffer.

According to another aspect of this invention, ° in a method for suppressing social and sexual behavior of domestic males. comprising administering the composition to a domestic animal, said composition comprising LHRH or LHRH. Also includes two or more different carriers individually conjugated to the analogue. A method is provided.

Another embodiment provides ovulation and estrous cyclicity in female livestock animals by administering the composition. Concerning methods for suppressing

Administration is parenteral, such as subcutaneous and/or intramuscular or intravenous injection, oral or can be absorbed through the skin or implanted within the animal or attached to the outside of the animal. It can be absorbed by the pump.

For cattle and horses, male calves are preferably immunized with the composition between 8 and 40 weeks of age. . For early horses, male lambs are preferably immunized at 8 to 24 weeks of age. booster note The injections are preferably given at 8 regular intervals, preferably with 1 to 3 booster injections. .

In the said method of this invention, within the scope of this method there is a Although the administration of substances (e.g., I, 'HRH) is discrete, the administration is done over relatively short intervals of time. This includes treatments performed over a period of time.

There are no obvious disadvantages compared to single administration of substances bound to at least two carriers. However, intervals of several minutes or hours usually give satisfactory results.

This invention is useful for immunocastration and ovariectomy of companion animals or farm animals, especially cats and dogs. It has special uses in Routine surgical procedures performed by veterinarians are expensive. Sticking, which can often cause trauma to the target animal and its owner; , at least other substances that elicit an immune response other than LHRH or LHRH analogs. is also conjugated to two different carriers, and then these conjugates are combined. An increase in the proportion of animals that respond may occur when the combination is administered to animals. immune response Examples of such substances that elicit responses include peptides and proteins, such as adrenocorticotropic hormones. Lumon, substance P1 human chorionic gonadotropin, somatostatin, epidermal and insulin-like growth factors, steroids such as androstenedione testosterone and other substances such as melatonin and prosthetics There is a Grandin.

In this specification, the following terms have the following meanings:

LHRH: Luteinizing hormone-releasing hormone is secreted from the pituitary gland. A specific substance required to stimulate luteinizing hormone and follicle-stimulating hormone .

Carrier: A substance that can elicit an immune response in animals when bound to a small substance. Especially proteins.

Antibody titer: 50% of 0.15 nCi” I-labeled LHRH was collected at 4°C for 16 hours. Dilution of antiserum to be combined. using polyethylene glycol with a final concentration of 14.5%. Achieved precipitation of gamma globulin.

DT...Diphtheria toxoid.

HSA...Human serum albumin.

KLH...Keyhole linbet hemocyanin.

TT...Tetanus toxoid.

CP...Corynebacterium parvum.

P, A,... Protein Carb from Staphylococcus aureus... - l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide.

Glut...Glutaraldehyde.

The following describes the invention by way of example only, giving experimental procedures, examples and tables. Make a note.

Figures 1 and 2 are for various treatment groups of male lambs and male calves, respectively. The antibody titer (logarithmic scale) is shown as a graph. Figures 3 and 4 are Nebacterium parvum (CP), keyhole linbet hemocyanin ( K L H), diphtheria toxoid (DT), tetanus toxoid (TT ) and these carriers bound to LHRH (CP, KLH, DT and and in Figure 3, when using the combination of KLHlTT and DT) Average testicular volume of male lambs is shown.

(A) Experimental procedure 1. Production of LHRH conjugate.

(a) Tetanus toxoid-LHRHLHRH (240gg) Tetanus ・Dissolve toxoid (35.5d, 248m9) in 20xQ distilled water and mix p) (adjusted to 6.0 with 0.1N NaOH. , 4 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide A freshly prepared solution containing ECDI hydrochloride (ECDI) was added dropwise and the reaction was allowed to stand at room temperature (2 2°C). The solution was dialyzed against 3 changes of saline (5 liters).

To assess the number of moles of LHRH bound to C. tetanus toxoid, 3 nCi 131■ labeled LHRH was added to the reaction mixture. Using this procedure, a wide range of The carrier protein was determined by counting the amount of “’I-LHRH” in the solution before and after dialysis. It is possible to measure the apparent binding of labeled LHRH with the substance. In this example, 39 mol of LHRH was bound to each mole of tetanus toxoid.

(b) LHRH-tetanus toxoid immunity using glutaraldehyde binding method Manufacture of epidemics.

10j! 9 amounts of LHRH and 1200 μQ (13,6 m9) of Tetanus ibis Soide was dissolved in 34xQ water and 0.1 mol NaOH was added to make the final p) (8 ．． It was set to 0. Then, glutaraldehyde (25% v/v of 4zQ) was added dropwise, The reaction was continued for 18 hours at 25°C. The solution was dialyzed as described in (a).

Calculating the “’I-LHRH activity before and after dialysis, it is found that 1 mole of tetanus toxoid A value of 38 moles of LHRH was obtained.

(c) Preparation of L) IRH-Protein A immunogen using carbodiimide coupling method .

LHRH (10x (1(1) mg of ff1 in water) was added to 5 ooμc of protein. A(50xy) and 10πQ were added to distilled water. 0.01 mol NaOH or Adjust p)1 to 6.5 with HCl2 and add 200z9 in 10zi2 of distilled water. ECD I was added. Incubate the reaction at 25°C for 18 hours as in section (a). I turned on.

When evaluating the incorporation of 1″I-LHRH into protein A, 3 changes of saline After dialysis against saline a LHRH/Protein A ratio of 1:36 was obtained.

(d) Preparation of LHRH-Protein A immunogen using glutaraldehyde binding method Construction.

10u amount of LHRH and 500 μm2 (5019) of protein A were added to 34zQ of distilled water and the pH was adjusted to 8 using 0.01 M NaOH. mixture Add 4xQ of 25% glutaraldehyde solution dropwise to the sample and let the solution stand at 25°C for 18 hours. Incubated. After dialysis against 3 exchanges of saline, as described above in section (A) The binding amount of protein A and LHRH was calculated using the iodine treatment LHRH method.

The results showed an apparent LHRH/Protein A ratio of 1=34.

(e) LHRH-keyhole limbus using carboxymid as the binding reagent Production of hemocyanin (KLH).

0.01 molar in 10i9 of LHRH and 20xy of KLH in 24m (l of distilled water) of HCl2 was added to give a final p)l of 6.5. Next, ECD I (20z Add 10s of 9/xQ solution + Q) to the mixture and incubate for 18 hours at 25°C. I turned on. As in section (a), the solution was dialyzed against 3 changes of saline. The above solution 1 to 1! Adding 'I-LHRH results in a LHRH/KLH model of 1 near 50 after dialysis. The ratio is shown below.

(f) Preparation of LHRH-KLH using glutaraldehyde as binder.

Dissolve 5η amounts of LHRH and lOO20KLH in 12xQ distilled water, and add 0.0 The pH was adjusted to 8.0 with 1M NaOH. Add 2 glutarua to this mixture. Hyde (25% w/v) was added dropwise and the solution was incubated for 18 hours at 25°C. Ta. The mixture was then dialyzed against 3 changes of saline as described in section (a). .

When adding “’I-LHRH to the solution, the obvious molar ratio of L　I　　/K　H　H showed that it was 849.

(g) Diphtheria toxoid-LHRHcLHRH (200r9) and diphtheria toxoid Terrier Toxoid (137+1, 219xy’) 10! Dissolve in Q distilled water ,0. The pH was adjusted to 6.0 with IN-NaOH.

The freshly prepared solution containing 0.69 ECDI was added dropwise to 2zQ distilled water and the reaction The reaction was allowed to proceed overnight at room temperature (22°C). Add the solution to 3 changes of saline (5 liters). and underwent dialysis.

(h) Corynebacterium parvum L) IRH.

LHRH (100m9) and Corynebacterium parvum (60R9) at 5 Dissolve zQ in distilled water and adjust p) to 6.0 using 0.1N NaOH. Ta. Add freshly prepared solution containing 0.29 ECDI dropwise to 2J112 distilled water. and incubate the mixture overnight at room temperature. N-LHRH.

LHRH (120g) and keyhole linbet hemocyanin (80m9 ) was dissolved in 10xQ distilled water, and p)( was dissolved using 0, I N-Na0H. It was adjusted to 6.0. A freshly prepared solution containing 0.49 ECDT in 4H distilled water. The liquid was added dropwise and the mixture was incubated overnight at room temperature. 3 changes of salt solution Dialyzed against water (5 liters).

To assess the effectiveness of binding, i.e. the moles of LHRH bound to the carrier protein, 0.15 nCi"J-LHRH was added to the above mixture. After each reaction, before and after dialysis. Percent binding "J-L) (RH) was calculated based on the values obtained. In other words, the effectiveness of the binding is good and 0. (B) Results Experiment 1 A flock of 42 male Merino lambs was used in the study.

The fish were selected according to live weight and divided into 7 groups consisting of 6 fish.

(However, during the experiment period, we lost several dogs. The cause of death was pneumonia.) Shown below.

[group] 1. Carrier protein KLH, protein A and tetanus in non-castrated fibers injected with toxoid.

A 26-week-old female was surgically castrated.

3. Vessels were immunized against LHRH-tetanus toxoid conjugate .

4. Vessels were immunized against LHRH-Protein A conjugate.

5. Vessels were immunized against the LHRH-KLH conjugate.

6. Cells were immunized against LHRH-HSA conjugate.

7. LHRH-TT, LHRH-Protein A and LHRH-KLH components immunized against the conjugate.

Doses, compositions and times of injection of the conjugate are shown in Table 1.

Each immunogen solution was emulsified with an equal volume of Freund's complete adjuvant. Then the male A mixture of 2x (l) was applied to four separate subcutaneous sites on the hind legs adjacent to the groin in lambs. was administered. 8j11 combined immunogen! injection using an emulsion of etc Booster injections were given with 50% Freund's incomplete adjuvant.

Blood samples were collected by venipuncture two weeks after the second booster injection. this Antibody titers in the experiments are shown in Table 3.

Experiment 2 A group of 35 male calves, primarily of the Friesian breed, was used in the experiment. calf The animals were fed skim milk, then weaned at 10 weeks of age and fed pasture. Biology as a basis for selection The bulls were divided into 5 groups of 7 animals using weights (3 bulls developed pneumonia during the experiment). (lost more). The treatment is shown below.

[Group] [Treatment] 1. Inject uncastrated male calf with carrier protein, tetanus toxoid, and keyhole. -Immunized against linbet hemocyanin and protein A (n=6) .

Table 1 Immunogen dose and booster schedule - LERR (Carb”) 400p g 400ug 400ug 400u9 Protein A- -XJml (Carb) 900ug 900ug 900s+g 900u g-kari” - around υ■ (Carb) 900ug 9001J (J 900L+9 9001J9H5 Enter-LERR (Carb) 5001g 500ug 500ug 500SJ9 Technus Toxoid-uU now with protein-IaEI + KLEI-IJR1! =te tanus toxoid -I-RRH (Carb) -400pg 400ug 400ug 400ug Contains protein -LIIRH (+job) 900IJg 900ug 900pg 5ou iKLE-Magazine (Carb) 9001g 900ug 900ug 900IJ9 Table 1 (Tsu ) T! L) Compositions for IRH immunization in boys of i, m and boo for immunogens Star Schedule 10 Weeks 18 Weeks 26 Weeks 34 Weeks Tetanus Kissoid ÷ KLI! + Protein included; tetanus toxoid 4001g 400SJ9 <OOyg 400LI9KLII 400L+9 <OOug 400u9 <00pg7' Roti 7A 900L+9 900 Mg　900Mg　900Mg2.A 14-week-old male calf was surgically castrated ( n=6).

3. Male calves were immunized against LHRH-ISA conjugate (n=6) .

4. - Immunization of male calves against LHRH-tetanus toxoid conjugate (n=6).

5. Male calf treated with tetanus toxoid, keyhole limpet hemocyanin and immunized against LHRH conjugated to protein A (n=6).

A summary of the composition, dose and immunization schedule for the experiment is shown in Table 2.

For the immunization procedure, prepare the immunogen solution using an equal volume of Freund's complete adjuvant. Emulsified. A total of 4-5 parts of 2 mQ emulsion solution in all individual treatment groups. injected at the position. For combined immunogen groups, 4-5 sites in male calves An emulsion solution of 81Q was injected into the patient. Subcutaneous injection near the front shoulder and chest of the calf I did it. For booster injections, administer the immunogen using Freund's incomplete adjuvant. emulsified. Blood samples were heparinized 2 weeks after the second booster injection. collected in a container. Antibody titers in this experiment are shown in Table 4.

The test was developed to study the social and sexual behavior of bulls. In this exam, Each male calf was individually exposed to a non-estrous female for 15 minutes to determine the social and sexual Behavior was observed and recorded.

The results of this test are shown in Table 5.

Table 2 after birth (14311 hours) (22a hours) (3o weeks) (38 weeks) - IJrRE (Carb) Association 360Mg 2oouq 2oou9 sooug-LERl l (Glut) membership fee 360Mg 200Mg 200Mg 5oouvKr J-W -(Carb) 360Mg 200Mg 2001Jg soow-(Glu t) 360s+g 200s+9 2001g 5001J9 protein λ -rJ1’l! I (Carb) 900Mg 650uq, 650$19 1 soou9-LIIIRI! (Glut) 900Mg 6SOug 650M g 1500pgT1S included-IJR1! -(Carb) 3sou9 200Mg 2oug 5oapy-(Glu t) 350S19 200SI9 200Mg 500u9 Tetanus toxo IF, #! J period + KLH-wy + Flotin A-IJIM: Tetanus toxoid 1'rxRB (Carb) 360M 200Mg zoou9 500119 Irokoro (Glut) 360$+9 200Mg 200Mg 500pgKL EI-uRlE -(Carb) 360ug 200ug 200ug 500u9-(Glu t) 360pg 200ug 200ug 500ug protein included -TXM (Carb) 90099 650ug 650ug 1500pg- ! JRH (Glut) 900ug 650ug 650pg 1500pg Knus Toxoid 1st Edition + Bu. Tin entry: Teta 57. -Toxoid 360u q zooug zoou9 5ouq-KIJI 360119 200u g 200SJ9 500149-7of<:zA 360SIg 200pg 　2001g00IJg ``Carbodiimide is used to bind LHRH and carrier protein.

Meeting: Glutaraldehyde is used to bind LHRH and carrier protein.

cormorant Table 5 LHRH versus social and sexual behavior of bulls after second booster injection Effect of immunization treatment The time required for the exam Percentage Uncastrated Neutered Combination TT HSA non-aggressive social Unisexual interaction 1.01 0.31 0.61 0.68 1.41 Sexual interaction Effects 0.61 0.09 0.39 0.48 0.90 Social and sexual Survey 0.72 0.31 0.35 0.46 0.70 Aggressive interaction 1.43 3.83 3.33 2.80 3.50 Various LHRH-conjugates male lambs and male lambs immunized by gates and their combinations, respectively. The antibody titers shown in Tables 3 and 4 for calves are graphed in Figures 1 and 2. to become a fu.

Figure 1 shows that, as expected, the assay system did not work in male lambs immunized with carrier protein alone. None of the lambs developed an immune response. Immunization with mono-LHRH conjugate In the case of lambs affected by the virus, variable results were obtained in both antibody titers and the number of animals responding. It was done.

In the case of KLH, only 40% of animals responded. The average titer for this response is 1:1 It is 30. Similar results were obtained for protein A, but 66% of the test animals showed an immune response and two animals died from pneumonia during the experiment. ), the flat of this response The average titer was 1:130. In the case of Tetanus toxoid (TT), a single compound The best results were obtained with the conjugate, with 60% of the animals showing an immune response and an average The value was 1:2200.

LHRH-TTSLHRH-Protein A and LHRH-KLH conjugates The results obtained for immunization with a mixture of All of the lambs tested showed a substantial immune response with an average titer of 1:5146. However, individual antibody titers varied within the range of 1:600 to 1:20,000. child These results are clearer than those obtained with single-LHRH-conjugates. significantly more favorable and statistically significantly superior (p <0.05).

Similar results with the same statistical significance as above are shown in FIG.

In Figure 2, Tetanus toxoid, Keyhole Limbet haemocyanii Male calves immunized with a mixture of protein A and LHRH combined with It showed a good immune response with a mean titer of 1:I70, which was associated with a single carrier protein. This was in contrast to the results obtained with conjugated LHRH.

The behavioral data presented in Table 5 shows that control animals were castrated, combined (LHRH to LH, TT and FA individually) and tetanus toxoid (TT to LH social and sexual behavior and social and show that it takes a lot of time in sexual research. As expected, castrated Males took the least amount of time for social and sexual behavior.

The time required for social and sexual behavior by bulls receiving the combination treatment is more than for objects, but less than for control animals. This is LHR We show that active immunization against H. can affect social and sexual behavior in bulls. show. Aggressive interactions in control animals than in castrated or combination-treated animals Although the time required for This was similar to the case for animals in the combined group (submissive test animals accompanied by aggressive interactions). it is possible to obtain). This also suggests that immunization may influence aggressiveness. Show that. Tetanus toxoid-L, HRH and HSA-LHRH con behavior of animals receiving the combination treatment than that of animals receiving Dinigate injections. It is clear that there was a significant impact on

Experiment 3 A flock of 80 Merino X Corizol lambs, 5 months old, was used in this experiment . Lambs (16 weeks old) were divided into 8 groups of 10 lambs each. 16 weeks for group 1 Animals were surgically castrated at the age of 10, and another group was left as unneutered. remaining 6 0 lambs received several LHRH antigen treatments.

Various LHRH-carrier protein conjugates were prepared as described above. used The antigens are diphtheria toxoid, tetanus toxoid, and keyhonil, respectively. - Separately binds to limpet hemocyanin and Corynebacterium parvum It was LHRH. The other two groups contain two different combinations of each LHRH complex. I was injected with water. The treatments are shown in Table 6, along with the total amount of conjugate used. . The mono-LHRH carrier protein in lamb neck with complete Freund's adjuvant. A subcutaneous injection was performed.

Eight weeks after the booster injection, a pen test was conducted on the animals to determine whether they were sexually active. We measured whether or not the In this study, each male was divided into four animals in an arena (51 x 51). The upper reproductive organs (anogenit) shown by the male were exposed to the female in heat for 15 minutes. al) The responses of the FJs, the occurrence of courtship behavior, the number of rides and the number of ejaculations were recorded. 6 Head males were observed simultaneously and all animals were observed on the same day. Animals 12 hours before pen testing were confined in the test arena and acclimatized to the test situation. Ride on the trial width and/or considered animals that ejaculated to be sexually active.

Table 6 L' = meaning and dose rate 1 10 Contrast 2　10　Castration 3 10 Garbage (resistant 1 yen) 4　10　pressure 4-[N]　1■ S10 Garbage-01 1■ 7　10　Pressure vessel-π◆Magazine 4-10 enclosure-mouth 3■8　10 Parts-door◆Carp: H +=−口　33Table Y Live weight change control in controls, castrated animals and treated juveniles 40.7 5 4.2 48.4 51.0 210.0 Castration 40.6 49.6 43.1 46.4 -L) IIRH-Tr 39.2 54.2 45.4 47.5 ? 46jLFE! IH-DT 39.3 53.9 45.6 48.7 B 3.3m-(? 39,3 55.8 46,8 49.3 I71.0Lhi 2) 1-KL) I 31! , Fu 55.3 46.3 4L Fu 153.3L) id-T+DT+KLH3B, °3 54, 7 46.5 4g, 6 F1. 0LFRH-DT+G'+KLH3B, 7 55.5 44.9 4L6 55 ．． 0 live weight and testicular size (measured using a series of measuring beads) every 4 weeks. It was measured.

against LHRH by both radioimmunoassay (RIA) and ELISA techniques. Antibody titers were measured. Plasma samples were soaked in phosphate buffer (0.05 mol, pH 7). , 6). Diluted sample (100μQ) with 100μQ bovine gamma Globulin (3% 9v/v) and 100μ5 of 2nCi”J-!, containing HRH Added to buffer. Tubes were incubated overnight at 4°C. 21% point in 111Q By adding lyethylene glycol 4000 and aspirating the supernatant after centrifugation, Free and bound L) (RH were separated.

Tubes were counted in a Clinigamma counter for 1 minute.

The live weights of all lambs are shown in Table 7. There were no significant differences between the groups, but Average live weight values for castrated lambs were higher than castrated males and lower than control lambs. each The mean testicular volumes across treatments are shown in Table 8 and Figures 3 and 4.

There was considerable variation for each antigen treatment. The maximum decrease in individual carriers is Observed in lambs immunized against LHRH combined with Pphtheria toxoid It was done. The next best carrier is tetanus toxoid, followed by keyhole toxoid. limpet hemocyanin and Corynebacterium parvum. 3 types Two different combinations of separate antigens have lower test values than each of the individual antigens. brought about.

As a result, a similar trend was observed in the RIA results. The median titer is It was the highest in the two groups using horsetail antigen, and whole limpet hemocyanin. , Corynebacterium parvum. RIA and ELISA IgG binding The results are shown in Table 9.

Table 3 Control 160.0 16g, 0 1113.5 187.0 210-0 castration --11- LK'JI-Tr 155.0 135.6 10+1.3 111.1 14 &, tLHRH-11yr 150.0 101. centre ? 3.3 59.1 B 3.3L) IPJ(-G'-144,0149,5162J 170.5 1 71.0 ratio 4-KIJi 153.3 133.5 136.1 127.2 151.3LH only)! -TT+DT+KLH13B, 9-! 11.1 62. 2 56.0? 1.0LH2H-DT+G'+KLH141,177,567 ,1411,355,0hehefNNINNN(NM MPIf'IMF'1MM P’lfQ #! Cry? seven network law Table 10 The effect on the proportion of animals that lost their shots Treatment group Ratio of ejaculated animals - TT 27g Go 4 Pressure 4 7 11g L” Jinji 4-Q’57ta Pressure 4-mouth 3/1° LFa) f -T F + DT + KLHO/10 Sho 4-Ba 10 G + Ero 0/1 In general, those lambs with high RIA titers are It had a similarly high titer. However, inconsistencies were observed in individual lambs; In some cases, high RIA titers may be observed in samples with low IgG titers. There was also.

These results indicate that RIA measurement is more specific than IgG ELISA assay. also indicates that multiple antibody subclasses are monitored.

The proportion of lambs in each group that were sexually active two months after the booster injection was Shown in Table 0. 10 out of a total of 10 lambs in the control group were sexually active. Ta. The best single antigen for 1 of 9 sexually active lambs was diphtheria. It was a toxoid. The order of effectiveness is that diphtheria toxoids, It was Corynebacterium parvum. Group 2 using the combination antigen showed no sexual activity. It showed no movement. L) Combination of IRH and TT, DT and KLH In the case of the 1,000 lambs that were fed the diet, none of the 10 lambs showed activity. Ta.

Similar results were obtained using different combinations of carrier, DT, CP and KLH. Again, a total of eight lambs (two lambs died during testing) showed activity. There were no lambs that did. None of the castrated lambs were sexually active. .

These results demonstrate that different combinations of carrier proteins present individual antigens separately. have been shown to produce a better physiological response than the This effect is caused by testicular rhinoplasty. observed in the proportion of animals that showed 20%, RIA titers, and sexual activity. this Overall, their results indicate that the combination of LHRH antigens is better than the individual antigens. It has been shown to produce an immune response.

Other aspects of this invention, as well as modifications and variations thereof, will be apparent to those skilled in the art from this specification. All such other aspects and modifications and variations thereof shall be apparent from reading the This is considered to be within the scope of the invention.

[References] Arimullah et al. (1974) - LH and anti-LHRH serum in rats Pre-ovulatory surge of FSH and interference with ovulation. Endophrynology, Volume 95, 32 Pages 3-325.

Tschitzpel et al. (1980) - Male rhesus on luteinizing hormone-releasing hormone Active immunization of monkeys, Biology Eve Reproduction, vol. 22, 333 -342 pages.

Clark et al. (1978) - Activity of fibers on luteinizing hormone-releasing hormone Immunization, and ovulation, and gonadotropins, prolactin and ovarian steroids Its effect on id secretion.

De U Cruz et al. (1976) - Antihematonic against luteinizing hormone-releasing hormone. Effect of serum administration on gonadal function during the estrous cycle in hamsters. endoph Rhinology, vol. 98, pp. 490-497.

Fraser et al. (1974) - Active immunization against luteinizing hormone-releasing hormone Treatment of serum and pituitary gonadotropins, testis and appendages in male rats. Effect on the vessel. Journal of Endophrynology, Volume 63, 399- 406 pages.

Fraser et al. (1978) - Immunization against luteinizing hormone-releasing hormone Ovarian changes in later rats. Journal of Endophrynology, 7 Volume 7, pages 85-93.

Fraser et al. (1982) - Progress Toward A Mer Contra 'Septive', (pp. 41-78, edited by Shefcourt and Sandra, M. John Willey & Sons, Bristol).

Barbert - “Veterinage Immunology” (Blackwell) (Sci. Entific, public).

Horazis and Byrne (1977) - for luteinizing hormone-releasing hormone Effect of immunization on reproduction in marmoset monkeys. Nature, vol. 265 , pp. 746-748.

(1982) - Male response to synthetic luteinizing hormone-releasing hormone Effects of active immunization of lambs and male calves.

Seriogenology, vol. 18, pp. 65-77.

Cock (1977) - ``Proceedings Eve the Fitz Inter National Combres Eve Endophrynology, (374-378 Page, edited by James, V.H.T., July 18-24).

McCormack et al. (1977) - Gonadotropin secretion in rhesus monkeys Effect of administration of luteinizing hormone-releasing hormone (LHRH) antiserum. Endofura Inology, Vol. 100, pp. 663-667.

21 Schlag and Frelag (1974) - Active immunization by testosterone Severe loss of sexual activity in treated rabbits. Experimenteia, 30 volumes , 434.435 pages.

Robertran et al. (1970) - Effects of weight loss on growth and carcass composition in beef cattle. effects of energy and dietary protein concentration. Journal Eve Agricultural Sci Enns (Camp Ridge, vol. 74, pp. 299-310).

Robert Ran et al. (1979) - Immunocastration in bulls. Beth Lev, Volume 105 , pp. 556-5'57 Robert Ran et al. (1981) - Immunization in bulls Further research on the dynamics. Beth Lev, vol. 111, pp. 529-531.

Robert Ran et al. (1984) - Immunocastration of young bulls for beef production. " "Manipulation Eve Growth in Farm Animals" (Roll) Shu and Dokalagan, pp. 137-145.

Schanbacher (1982) - Testosterone and luteinizing hormone release hormone Response of male lambs to active immunization against Lumon. journal eve fi Geology, 242e, pp. 201-205.

Schanbacher (1984) - Active immunization against LHRH in males. " Immunological Aspects Eve Reproduction in Mammals” . pp. 345-362, edited by Clifton. Butterworth.

fresh treatment Figure 1 Antibody titer in male medium Place 22n Figure 2 Time since first injection (months) Time since first injection (months) international search report

Claims (10)

[Claims] (1) A composition for the purpose of producing antibodies specific to LHRH in animals. LHRH or A composition comprising two or more different carriers individually conjugated to analogs of LHRH. composition. (2) The composition according to claim 1, wherein at least two types of carriers are carrier proteins. thing. (3) A claim in which at least one of the carrier proteins is diphtheria toxoid. The composition according to item 2. (4) Any one of claims 1 to 3, wherein the composition further comprises an adjuvant. Compositions as described in Section. (5) the composition contains an adjuvant selected so that it does not cause inflammation; The composition according to claim 4. (6) A male home comprising administering the composition according to any one of claims 1 to 5. How to control social and sexual behavior in livestock. (7) A female home comprising administering the composition according to any one of claims 1 to 5. A method for suppressing ovulation and estrous cycle in livestock. (8) The method according to claim 6, wherein the composition is administered twice or more. . (9) The animal is selected from dogs, cats, cows, sheep, donkeys, rabbits, and deer. 9. A method according to any one of claims 6-8, wherein: (10) Can elicit an immune response in animals when conjugated with a carrier A composition intended for the production of a substance-specific antibody in an animal, the composition comprising at least A composition comprising a mixture of substances conjugated to two different carriers.